Non-peptide macrocyclic histone deacetylese (HDAC) inhibitors and methods of making and using thereof

ABSTRACT

Compounds of Formula I or II, and methods of making and using thereof, are described herein. 
     
       
         
         
             
             
         
       
         
         
           
             M represents a macrolide subunit, 
             E is a C 1-6  group, optionally containing one or more heteroatoms, 
             D is an alkyl or aryl group, 
             A is a linking group connected to D, 
             B is an alkyl, alkylaryl or alkylheteroaryl spacer group, 
             ZBG is a Zinc Binding Group, 
             R 1 , R 2  and R 4  are independently are selected from hydrogen, a C1-6 alkyl group, a C 2-6  alkenyl group, a C 2-6  alkynyl group, a C 1-6  alkanoate group, a C 2-6  carbamate group, a C 2-6  carbonate group, a C 2-6  carbamate group, or a C 2-6  thiocarbamate group, 
             R 3  is hydrogen or —OR 5 , 
             R 5  is selected from a group consisting of Hydrogen, a C 1-6  alkyl group, a C 2-6  alkenyl group, a C 2-6  alkynyl group, C 1-6  alkanoate group, C 2-6  carbamate group, C 2-6  carbonate group, C 2-6  carbamate group, or C 2-6  thiocarbamate group.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 12/643,633filed Dec. 21, 2009, now U.S. Pat. No. 8,188,054, issued May 9, 2012,which is a continuation-in-part of PCT/US2008/068787 filed Jun. 30,2008, which claims priority to U.S. Ser. No. 60/947,036 filed Jun. 29,2007.

FIELD OF THE INVENTION

The present invention generally relates to non-peptide macrocyclichistone deacetylase (HDAC) inhibitors and methods of making and usingthereof.

BACKGROUND OF THE INVENTION

Histone deacetylases (HDACs) and histone acetyltransferases (HATs) aretwo functionally opposing enzymes which tightly regulate chromatinstructure and function by maintaining the equilibrium between theacetylated- and deacetylated-states of nucleosomal histones. Aberrationsin intracellular histone acetylation-deacetylation equilibrium have beenlinked to the repression of a subset of genes resulting in excessiveproliferation and are implicated in a number of malignant diseases(Jenuwein, T.; Allis, C. D., Science 293, 1074-1080 (2001); Marks, P.;Rifkind, R. A.; Richon, V. M.; Breslow, R.; Miller, T.; Kelly, W. K.,Nat. Rev. Cancer, 1, 194-202 (2001)). HDACs function as part ofmultiprotein complexes that catalyze the removal of acetyl groups fromthe ε-amino groups of specific lysine residues located near theN-termini of nucleosomal core histones (Grozinger, C. M.; Schreiber, S.L., Chem. Biol. 9, 3-16 (2002)). HDAC-catalyzed deacetylation results inpositively charged, hypoacetylated histones which bind tightly to thephosphate backbone of DNA, thus inducing gene-specific repression oftranscription. Inhibition of HDAC function results in the weakening ofthe bond between histones and DNA, thus increasing DNA accessibility andgene transcription.

Eighteen distinct human HDACs have been identified to date. They areclassified into three major families based on their homology to threeSaccharomyces cerevisiae HDACs: RPD3, HDA1, and SIR2. Class I includesHDACs 1, 2, 3 and 8. Class II includes HDACs 4, 5, 6, 7, 9, 10 and 11.The third class of HDACs is the sirtuins, which are homologicallydistinct from all the currently known HDACs. HDAC inhibition by smallmolecules has been observed for the natural product (R)-trichostatin Awhich induced cell differentiation of murine erythroleukemia cells andhyperacetylation of histone proteins at nanomolar concentrations.Suberoylanilide hydroxamic acid (SAHA) has also been identified as aHDAC inhibitor.

Inhibition of HDACs is an emerging therapeutic strategy in cancertherapy. HDAC inhibitors have demonstrated ability to arrestproliferation of nearly all transformed cell types, including epithelial(melanoma, lung, breast, pancreas, ovary, prostate, colon and bladder)and hematological (lymphoma, leukemia and multiple myeloma) tumors(Kelly, W. K; O'Connor, O. A.; Marks, P. A., Expert. Opin. Investig.Drugs, 11, 1695-1713 (2002)). Additionally, HDAC inhibitors havedemonstrated other biological activity including anti-inflammatory,anti-arthritic, anti-infective, anti-malarial, cytoprotective,neuroprotective, chemopreventive and/or cognitive enhancing effects.

All HDAC inhibitors so far reported typically fit a three-motifpharmacophoric model namely, a zinc-binding group (ZBG), a hydrophobiclinker and a recognition cap-group (Miller, T. A.; Witter, D. J.;Belvedere, S., J. Med Chem., 46, 5097-5116 (2003)). Structuralmodifications of the ZBG yielding hydroxamate isosteres such asbenzamide, α-ketoesters, electrophilic ketones, mercaptoamide andphosphonates have been reported. The cap-group may present opportunitiesto discover more potent and/or selective HDAC inhibitors. Toward thisend, recent work by Schreiber and co-workers has led to theidentification of cap group-modified agents that display differentialinhibition against specific HDAC sub-types (Wong, J.; Hong, R.;Schreiber, S., J. Am. Chem. Soc. 125, 5586-5587 (2003); Haggarty, S. J.;Koeller, K. M.; Wong, J. C.; Grozinger, C. M.; Schreiber, S. L., Proc.Natl. Acad. Sci. USA, 100, 4389-4394 (2003)).

Cyclic-peptide moieties are the most complex of all HDAC inhibitorcap-groups and present an opportunity for the modulation of thebiological activities of HDAC inhibitors. The macrocycle group is madeup of hydrophobic amino acids and the prominent difference among themembers of this class is in the amino acid side-chain substitution onthe ring. Mechanistically, cyclic-peptide HDAC inhibitors can be dividedinto two classes: (i) reversible HDAC inhibitors and (ii) irreversibleHDAC inhibitors, due to the alkylative modification of HDAC enzyme bythe epoxy-ketone moiety on their side-chain. HDAC inhibitory activityand selectivity can vary significantly by changing the side-chain ofeach amino acid and/or the pattern of the combination of amino acidchirality.

Although cyclic-peptide HDAC inhibitors may possess potent HDACinhibitory activity, their broad application in specific therapies, suchas cancer therapy, currently remains largely unproven. The absence ofclinically effective cyclic-peptide HDAC inhibitors may be in part dueto development problems characteristic of large peptides, particularlypoor oral bioavailability. In fact, the overall in vivo efficacy ofcyclic-peptide HDAC inhibitors is complicated by their membranepenetration ability. HDAC inhibitory potency has been noted to increasewith increase in the hydrophobicity of the macrocyclic ring (Meinke, P.T.; Liberator, P., Curr. Med. Chem., 8, 211-235 (2001)). Unfortunately,SAR studies for this class of compounds have been impaired largelybecause most macrocyclic HDAC inhibitors known to date contain peptidemacrocycles. In addition to retaining the pharmacologicallydisadvantaged peptidyl-backbone, they offer only limited opportunity forside-chain modifications.

To date, several other structurally distinct small molecule HDACinhibitors have been reported including hydroxamates, benzamides,short-chain fatty acids, electrophilic ketones and cyclic-peptides(Miller, T. A.; Witter, D. J.; Belvedere, S., J. Med. Chem. 46,5097-5116 (2003); Rosato, R. R.; Grant, S., Expert Opin. Invest. Drugs,13, 21-38 (2004); Monneret, C., Eur. J. of Med. Chem., 40, 1-13 (2005);Yoo, C. B.; Jones, P. A., Nature Reviews Drug Discovery, 5, 37-50(2006)). Most of these agents have been shown to non-selectively inhibitthe deacetylase activity of class I/II HDAC enzymes. The HDAC inhibitorSAHA has been approved by the FDA for the treatment of cutaneous T celllymphoma. However, a large number of the identified HDAC inhibitors haveelicited only limited in vivo antitumor activities and have notprogressed beyond preclinical characterizations.

Therefore, there is a need to develop new HDAC inhibitors with improvedefficacy, and better pharmacokinetic properties for use as therapeuticagents, such as anti-cancer agents and anti-parasitic agents.

It is therefore an object of the invention to provide non-peptidemacrocyclic HDAC inhibitors having improved efficacy and methods ofmaking and using thereof.

SUMMARY OF THE INVENTION

Compounds of Formula I and II, and methods of making and using thereof,are described herein.

wherein M represents a macrolide subunit,

E is a substituted or unsubstituted C₁₋₆ group, optionally containingone or more heteroatoms, wherein the carbon atoms and/or heteroatoms arein a linear and/or cyclic arrangement,

D is a substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl group,

A is a linking group connected to D,

B is an alkyl, heteroalkyl, alkylaryl or alkylheteroaryl spacer group,

ZBG is a Zinc Binding Group,

R₁₃ is selected from hydrogen, a substituted or unsubstituted C₁₋₆ alkylgroup, a substituted or unsubstituted C₂₋₆ alkenyl group, a substitutedor unsubstituted C₂₋₆ alkynyl group, a substituted or unsubstitutedalkanoate group, a substituted or unsubstituted C₂₋₆ carbamate group, asubstituted or unsubstituted C₂₋₆ carbonate group, a substituted orunsubstituted C₂₋₆ carbamate group, or a substituted or unsubstitutedC₂₋₆ thiocarbamate group,

R₁₄ and R₁₆ is selected from hydrogen, hydroxyl, a substituted orunsubstituted C₁₋₆ alkyl group, a substituted or unsubstituted C₂₋₆alkenyl group, a substituted or unsubstituted C₂₋₆ alkynyl group, asubstituted or unsubstituted C₁₋₆ alkanoate group, a substituted orunsubstituted C₂₋₆ carbamate group, a substituted or unsubstituted C₂₋₆carbonate group, a substituted or unsubstituted C₂₋₆ carbamate group, ora substituted or unsubstituted C₂₋₆ thiocarbamate group,

R₁₅ is hydrogen or —OR₁₇,

R₁₇ is selected from a group consisting of hydrogen, a substituted orunsubstituted C₁₋₆ alkyl group, a substituted or unsubstituted C₂₋₆alkenyl group, a substituted or unsubstituted C₂₋₆ alkynyl group, asubstituted or unsubstituted C₁₋₆ alkanoate group, a substituted orunsubstituted C₂₋₆ carbamate group, a substituted or unsubstituted C₂₋₆carbonate group, a substituted or unsubstituted C₂₋₆ carbamate group, ora substituted or unsubstituted C₂₋₆ thiocarbamate group.

The macrolide subunit can be attached to the pyran moiety at multiplelocations on the macrolide subunit. In one embodiment, the compoundcontains the macrolide M9, M27, M28, M29, or M30 in Table 2, z is 1, nis 1, D is an aryl group, such as a phenyl group, A is a 1,2,3-triazolylgroup, B is an alkyl group having from 5-8 carbons, and ZBG is ahydroxamate group.

The compounds can be administered as the free acid or base, or as apharmaceutically acceptable salt, prodrug, or solvate. The compounds canbe formulated with a pharmaceutically acceptable carrier and, optionallyone or more pharmaceutically acceptable excipients, for enteral,parenteral, or topical administration. The compounds can be formulatedfor immediate release and/or controlled release. Examples of controlledrelease formulations include sustained release, delayed release,pulsatile release, and combinations thereof.

The compounds may be useful as anti-cancer agents, anti-inflammatoryagents, anti-infective agents, anti-parasitic agents, such asanti-malarial agents and antileishmanial agents, cytoprotective agents,chemopreventive agents, prokinetic agents, and/or cognitive enhancingagents. The presence of the macrolide group may allow for the targeteddelivery of the HDAC inhibitor in view on the ability of macrolides toaccumulate in specific tissues.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Macrolide”, as used herein, includes, but is not limited to,multi-member lactonic ring molecules, wherein “member” refers to thecarbon atoms and heteroatoms in the ring, and “multi” is a numbergreater than about 10, preferably from 10 to about 20, more preferably12-, 14-, 15-, 16-, 17- or 18-member lactonic rings. Suitable macrolidesinclude, but are not limited to, azithromycin and its derivatives;clarithromycin and its derivatives; erythromycin and its derivatives;bridged bicyclic macrolides, such as EDP-420 and its derivatives;dirithromycin and its derivatives,9-dihydro-9-deoxo-9a-aza-9a-homoerythrornycin and its derivatives; HMR3004 and its derivatives, HMR 3647 and its derivatives; HMR 3787 and itsderivatives; josamycin and its derivatives; erythromycylamine and itsderivatives; ABT 773 and its derivatives; TE 802 and its derivatives;flurithromycin and its derivatives; tylosin and its derivatives;tilmicosin and its derivatives; oleandomycin and its derivatives;desmycosin and its derivatives; CP-163505 and its derivatives; EDP-420and its derivatives; roxithromycin and its derivatives; miocamycin andits derivatives; rokitamycin and derivatives thereof, such as ketolides(e.g., 3-ketone), lactams (e.g., 8a- or 9a-lactams) and derivativeslacking one or more sugar moieties.

“Aryl”, as used herein, refers to 5-12-membered, preferably 5-, 6- and7-membered aromatic, heterocyclic, fused aromatic, fused heterocyclic,biaromatic, or bihetereocyclic ring systems, optionally substituted, forexample, by halogens, alkyl-, alkenyl-, and alkynyl-groups. Broadlydefined, “Ar”, as used herein, includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “Ar” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (i.e.,“fused rings”) wherein at least one of the rings is aromatic, e.g., theother cyclic ring or rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocycles. Examples of heterocyclic ringinclude, but are not limited to, benzimidazolyl, benzofuranyl,benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl,carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, (uranyl,furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl,isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl,phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl,phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl and xanthenyl.

“Zinc binding group” or “ZBG”, as used herein, refers to a moiety ormoieties capable of inhibiting the activity of zinc metalloenzymesincluding, but not limited to, HDAC and matrix metalloproteinase (MMP)activity. Suitable examples include, but are not limited to,hydroxamates, N-formyl hydroxylamine (or retro-hydroxamate),carboxylates, thiols, dithiols, trithiocarbonates, thioesters,benzamide, keto groups, mercaptoacetamides, 2-ketoamides, epoxides,epoxyketones, trifluoromethyl ketones, hydroxypyridinones, such as2-hydroxy and 3-hydroxypyridinones, pyrones, hydroxylpyridinethiones,such as 3-hydroxy-2-methylpyridine-4-thione, and thiopyrones.

“Alkyl”, as used herein, refers to the radical of saturated orunsaturated aliphatic groups, including straight-chain alkyl, alkenyl,or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups,cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkylsubstituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, andcycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unlessotherwise indicated, a straight chain or branched chain alkyl has 30 orfewer carbon atoms in its backbone (e.g., C1-C30 for straight chain,C3-C30 for branched chain), preferably 20 or fewer, more preferably 10or fewer, most preferably 6 or less. Likewise, preferred cycloalkylshave from 3-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure. “Heteroalkyl”, as usedherein, refers to an alkyl group containing one or more heteroatoms,such as O, S, or N.

“Alkoxycarbonyl”, as used herein, refers to a substituent having thefollowing chemical formula:

wherein R is a linear, branched, or cyclic alkyl group, wherein j isfrom about 1 to about 12,

“Alkoxycarbamido”, as used herein, refers to a substituent having thefollowing chemical formula:

wherein R₈ is alkoxy and R₉ is hydrogen, alkoxy-alkyl, or alkanoyl, andj is from about 1 to about 12.

“Alkylaryl”, as used herein, refers to an alkyl group substituted withan aryl group (e.g., an aromatic or hetero aromatic group).

“Heterocycle” or “heterocyclic”, as used herein, refers to a cyclicradical attached via a ring carbon or nitrogen of a monocyclic orbicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ringatoms, consisting of carbon and one to four heteroatoms each selectedfrom the group consisting of non-peroxide oxygen, sulfur, and N(Y)wherein Y is absent or is H, O, (C₁₋₄)alkyl, phenyl or benzyl, andoptionally containing 1-3 double bonds and optionally substituted withone or more substituents. Examples of heterocyclic ring include, but arenot limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, (uranyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

“Heteroaryl”, as used herein, refers to a monocyclic aromatic ringcontaining five or six ring atoms consisting of carbon and 1, 2, 3, or 4heteroatoms each selected from the group consisting of non-peroxideoxygen, sulfur, and N(Y) where Y is absent or is H, O, (C₁-C₈) alkyl,phenyl or benzyl. Non-limiting examples of heteroaryl groups includefuryl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl,isothiazoyl, pyrazolyl, pyrazinyl, tetrazolyl, pyridyl, (or itsN-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl(or its N-oxide), quinolyl (or its N-oxide) and the like. The term“heteroaryl” can include radicals of an ortho-fused bicyclic heterocycleof about eight to ten ring atoms derived therefrom, particularly abenz-derivative or one derived by fusing a propylene, trimethylene, ortetramethylene diradical thereto. Examples of heteroaryl can be furyl,imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl,isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl (or itsN-oxide), thientyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl(or its N-oxide), quinolyl (or its N-oxide), and the like.

“Halogen”, as used herein, refers to fluorine, chlorine, bromine, oriodine.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

“Substituted”, as used herein, means that the functional group containsone or more substituents including, but not limited to, hydroxyl,halogen, nitro, aldehyde, ketone, carboxylic acid, ester, ether, amino,alkyl amino, dialkyl amino, amide, thiol, thioether, thione, sulfate,phosphate, and combinations thereof.

“Pharmaceutically acceptable salt”, as used herein, refer to derivativesof the compounds defined by Formula I and II wherein the parent compoundis modified by making acid or base salts thereof. Example ofpharmaceutically acceptable salts include but are not limited to mineralor organic acid salts of basic residues such as amines; and alkali ororganic salts of acidic residues such as carboxylic acids. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. Suchconventional non-toxic salts include those derived from inorganic acidssuch as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, andnitric acids; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,tolunesulfonic, naphthalenesulfonic, methanesulfonic, ethane disulfonic,oxalic, and isethionic salts.

The pharmaceutically acceptable salts of the compounds can besynthesized from the parent compound, which contains a basic or acidicmoiety, by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000, p. 704; and “Handbook of Pharmaceutical Salts:Properties, Selection, and Use,” P. Heinrich Stahl and Camille G.Wermuth, Eds., Wiley-VCH, Weinheim, 2002.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio.

“Prodrug”, as used herein, refers to a pharmacological substance (drug)which is administered in an inactive (or significantly less active)form. Once administered, the prodrug is metabolized in the body (invivo) into the active compound.

“Solvate”, as used herein, refers to a compound which is formed by theinteraction of molecules of a solute with molecules of a solvent.

“Reverse ester”, as used herein, refers to the interchange of thepositions of the oxygen and carbon groups in a series of structurallyrelated compounds

“Reverse amide”, as used herein, refers to the interchange of thepositions of the nitrogen and carbon groups in a series of structurallyrelated compounds.

II. Compounds

Compounds of Formula I or II, and methods of making and using thereof,are described herein.

wherein M represents a macrolide subunit,

E is a substituted or unsubstituted C₁₋₆ group, optionally containingone or more heteroatoms, wherein the carbon atoms and/or heteroatoms arein a linear and/or cyclic arrangement,

D is a substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl group,

A is a linking group connected to D,

B is an alkyl, heteroalkyl, alkylaryl or alkylheteroaryl spacer group,

ZBG is a Zinc Binding Group,

R₁₃ is selected from hydrogen, a substituted or unsubstituted C₁₋₆ alkylgroup, a substituted or unsubstituted C₂₋₆ alkenyl group, a substitutedor unsubstituted C₂₋₆ alkynyl group, a substituted or unsubstituted C₁₋₆alkanoate group, a substituted or unsubstituted C₂₋₆ carbamate group, asubstituted or unsubstituted C₂₋₆ carbonate group, a substituted orunsubstituted C₂₋₆ carbamate group, or a substituted or unsubstitutedC₂₋₆ thiocarbamate group,

R₁₄ and R₁₆ is selected from hydrogen, hydroxyl, a substituted orunsubstituted C₁₋₆ alkyl group, a substituted or unsubstituted C₂₋₆alkenyl group, a substituted or unsubstituted C₂₋₆ alkynyl group, asubstituted or unsubstituted C₁₋₆ alkanoate group, a substituted orunsubstituted C₂₋₆ carbamate group, a substituted or unsubstituted C₂₋₆carbonate group, a substituted or unsubstituted C₂₋₆ carbamate group, ora substituted or unsubstituted C₂₋₆ thiocarbamate group,

R₁₅ is hydrogen or —OR₁₇,

R₁₇ is selected from a group consisting of hydrogen, a substituted orunsubstituted C₁₋₆ alkyl group, a substituted or unsubstituted C₂₋₆alkenyl group, a substituted or unsubstituted C₂₋₆ alkynyl group, asubstituted or unsubstituted C₁₋₆ alkanoate group, a substituted orunsubstituted C₂₋₆ carbamate group, a substituted or unsubstituted C₂₋₆carbonate group, a substituted or unsubstituted C₂₋₆ carbamate group, ora substituted or unsubstituted C₂₋₆ thiocarbamate group.

Examples of the linking group A include, but are not limited to, amide,reverse amide, ester, reverse ester, alkoxyl, sulfonyl, sulfonyl,sulfonyl, sulfonamido, ketone, sp³ hybridized carbon, sp² hybridizedcarbon, sp hybridized carbon, 5 or 6 membered heterocyclic ringsincluding but not limiting to 1,2,3-triazolyl, 1,2,4-triazolyl,1-tetrazolyl, 1-indolyl, 1-indazolyl, 2-isoindolyl, 7-oxo-2-isoimdolyl,1-pirinyl, 3-isothiazolyl, 4-isothiazolyl and 5-isothiazolyl,1,3,4,-oxadiazole, 4-oxo-2-thiazolinyl or5-methyl-1,3,4-thiadiazol-2-yI, thiazoledione, 1,2,3,4-thiatriazole,1,2,4-dithiazolone, pyridine, thiophene, furan, pyrazoline, pyrimidine,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 3-pyrazolyl, 2-quinolyl,3-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 2-quinazolinyl,4-quinazolinyl, 2-quinoxalinyl, 1-phthalazinyl, 4-oxo-2-imidazolyl,2-imidazolyl, 4-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl,3-pyrazolyl 4-pyrazolyl, 5-pyrazolyl, 2-oxazolyl, 4-oxazolyl,4-oxo-2-oxazolyl, 5-oxazolyl, 4,5-dihydrooxazole, 1,2,3-oxathiole,1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole,2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 3-isothiazole, 4-isothiazole,5-isothiazole, 2-indolyl, 3-indolyl, 3-indazolyl, 2-benzoxazolyl,2-benzothiazolyl, 2-benzimidazolyl, 2-benzofuranyl, 3-benzofuranyl,benzoisothiazole. benzisoxazole, 2-furanyl, 3-furanyl, 2-thienyl,3-thienyl, 2-pyrrolyl, pyrrolyl, 3-pyrrolyl, 3-isopyrrolyl,4-isopyrrolyl, 5-isopyrrolyl, 1,2,3-oxathiazole-1-oxide,1,2,4-oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl, 5-oxo-1,2,4-oxadiazol-3-yl,1,2,4-thiadiazol-3-yl, 1,2,3-thiadiazol-5-yl,3-oxo-1,2,3-thiadiazol-5-yl, 1,3,4-thiadiazol-5-yl,2-oxo-1,3,4-thiadiazol-5-yl, 1,2,4-triazol-3-yl, 1,2,4-triazol-5-yl,1,2,3,4-tetrazol-5-yl, 5-oxazolyl, 1-pyrrolyl, 1-pyrazolyl. Each ofthese moieties may be substituted as appropriate.

B is a substituted or unsubstituted alkyl, alkylaryl or alkylheteroarylspacer group. Suitable alkyl spacer group chain length ranges from aboutC₄ to about C₁₂, optionally substituted by one or more double and/ortriple bonds. The total number of atoms in the alkylaryl andalkylheteroaryl groups is from about 6 to about 50, preferably fromabout 6 to about 30, more preferably from about 6 to about 20, mostpreferably from about 6 to about 10.

M is a macrolide subunit. Suitable macrolide subunits include, but arenot limited to, multi-member lactonic ring molecules, wherein “member”refers to the carbon atoms or heteroatoms in the ring, and “multi” is anumber greater than about 10, preferably from 10 to about 20, morepreferably 12-, 14-, 15-, 16, 17-, or 18-member lactonic rings.Exemplary macrolides include, but are not limited to, azithromycin andits derivatives; clarithromycin and its derivatives; erythromycin andits derivatives; bridged bicyclic macrolides, such as EDP-420 and itsderivatives; dirithromycin and its derivatives,9-dihydro-9-deoxo-9a-aza-9a-homoerythromycin and its derivatives; HMR3004 and its derivatives, HMR 3647 and its derivatives; HMR 3787 and itsderivatives; josamycin and its derivatives; erythromycylamine and itsderivatives; ABT 773 and its derivatives; TE 802 and its derivatives;flurithromycin and its derivatives; tylosin and its derivatives;tilmicosin and its derivatives; oleandomycin and its derivatives;desmycosin and its derivatives; CP-163505 and its derivatives; EDP-420and its derivatives; roxithromycin and its derivatives; miocamycin andits derivatives; rokitamycin and derivatives thereof, such as ketolides(e.g., 3-ketone), lactams (e.g., 8a- or 9a-lactams) and derivativeslacking one or more sugar moieties.

In one embodiment, the macrolide M has the formula:

as described in U.S. Patent Application Publication No. 2007/0149463,which is incorporated herein by reference, wherein

W is selected from —C(O)—, —C(═NOR₁₁)—, —CH(—OR₁₁)—, —NR₁₁CH₂—,—CH₂NR₁₁—, —CH(NR₁₁R₁₁)—, —C(═NNR₁₁R₁₁)—, —NR₁₁C(O)—, —C(O)NR₁₁—, and—C(═NR₁₁)—;

R is selected from the group consisting of H and C₁₋₆ alkyl;

R₁ is selected from the group H, halogen, —NR₁₁R₁₁, NR₁₁C(O)R₁₁, —OR₁₁,—OC(O)R₁₁, —OC(O)OR₁₁, —OC(O)NR₁₁R₁₁, —OC₁₋₆alcyl-R₁₂, —OC(O)C₁₋₆alkyl-R₁₂, —OC(O)OC₁₋₆ alkyl-R₁₂, —OC(O)NR₁₁C₁₋₆ alkyl-R₁₂, C₁₋₆ alkyl,C₁₋₆ alkenyl, C₁₋₆ alkynyl, optionally is substituted with one or moreR₁₂ groups;

R₂ is H;

R₃ is selected from H, —OR₁₁, —OC₁₋₆ alkyl-R₁₂, —OC(O)R₁₁, —OC(O)C₁₋₆alkyl-R₁₂, —OC(O)OR₁₁, —OC(O)OC₁₋₆ alkyl-R₁₂, —OC(O)NR₁₁R₁₁,—OC(O)NR₁₁C₁₋₆ alkyl-R₁₂; alternatively, R₃ is a pyran ring which can besubstituted as defined above in Formulae I and II;

R₄ is selected from H, R₁₁, —C(O)R₁₁, —C(O)OR₁₁, —C(O)NR₁₁R₁₁, —C₁₋₆alkyl-T-R₁₁, —C₂₋₆ alkenyl-T-R₁₁, and —C₂₋₆ alkynyl-T-R₁₁; alternativelyR₃ and R₄ taken together form

T is selected from —C(O)—, —C(O)O—, —C(O)NR₁₁—, —C(═NR₁₁)—,—C—(═NR₁₁)O—, —C(═NR₁₁)NR₁₁—, g) —OC(O)—, —OC(O)O—, —OC(O)NR₁₁—,—NR₁₁C(O)—, —NR₁₁C(O)O—, —NR₁₁C(O)NR₁₁—, —NR₁₁C(═NR₁₁)NR₁₁—, and—S(O)_(p)—, wherein p 0-2;

R₅ is selected from R₁₁, —OR₁₁, —NR₁₁R₁₁, —OC₁₋₆ alkyl-R₁₂, —C(O)R₁₁,—C(O)C₁₋₆ alkyl-R₁₂, —OC(O)R₁₁, —OC(O)G₁₋₆ alkyl-R₁₂, —OC(O)OR₁₁,—OC(O)OC₁₋₆ alkyl-R₁₂, —OC(O)NR₁₁R₁₁, —OC(O)NR₁₁C₁₋₆ alkyl-R₁₂,—C(O)C₂₋₆ alkenyl-R₁₂, and —C(O)C₂₋₆ alkynyl-R₁₂;

alternatively, R₄ and R₅ taken together with the atoms to which they arebonded, form:

wherein, Q is CH or N, and R₂₃ is —OR₁₁ or R₁₁;

R₆ is selected from —OR₁₁, alkoxy-R₁₂, —C(O)R₁₁, —OC(O)R₁₁, —OC(O)OR₁₁,—OC(O)NR₁₁R₁₁, NR₁₁R₁₁;

alternatively, R₅ and R₆ taken together with the atoms to which they areattached form a 5-membered ring by attachment to each other through alinker selected from —OC(R₁₂)₂O—, —OC(O)O—, —OC(O)NR₁₁—, d) —NR₁₁C(O)O—,—OC(O)NOR₁₁—, —NOR₁₁C(O)O—, —OC(O)NNR₁₁R₁₁—, —NNR₁₁R₁₁C(O)O—,—OC(O)C(R₁₂)₂—, —C(R₁₂)₂C(O)O—, —OC(S)O—, —OC(S)NR₁₁—, —NR.₁₁C(S)O—, n)—OC(S)NOR₁₁—, —NOR₁₁C(S)O—, —OC(S)NNR₁₁R₁₁—, —NNR₁₁R₁₁C(S)O—,—OC(S)C(R₁₂)₂—, —C(R₁₂)₂C(S)O—;

alternatively, W, R₅, and R₆ taken together with the atoms to which theyare attached form:

wherein J is selected from the group consisting of O, S, and NR₁₁;

R_(6′) is selected from H, unsubstituted or substituted C₁₋₄ alkyl, C₂₋₄alkenyl, which can be further substituted with C₁₋₂ alkyl or one or morehalogens, C₂₋₄ alkynyl, which can be further substituted with C₁₋₂alicyl or one or more halogens, aryl or heteroaryl, which can be furthersubstituted with C₁₋₂ alkyl or one or more halogens, —C(O)H, —COOH, —CN,—COOR₁₁, —C(O)NR₁₁R₁₁, —C(O)R₁₁, —C(O)SR₁₁;

alternatively R₆ and R_(6′) taken together with the atom to which theyare attached to form an epoxide, a carbonyl, an olefin, or a substitutedolefin, or a C₃-C₇ carbocyclic, carbonate, or carbamate, wherein thenitrogen of the carbamate can be further substituted with a C₁-C₆ alkyl;

R₇ is selected from C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl,optionally is substituted with one or more R₁₂ groups;

R₁₁, for each occurrence, is selected from H, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, C₆₋₁₀ saturated, unsaturated, or aromatic carbocycle, 3-12membered saturated, unsaturated, or aromatic heterocycle containing oneor more heteroatoms selected from nitrogen, oxygen, and sulfur,—C(O)—C₁₋₆ alkyl, —C(O)—C₂₋₆ alkenyl, —C(O)—C₂₋₆ alkynyl, —C(O)—C₆₋₁₀saturated, unsaturated or aromatic carbocycle, —C(O)-3-12 memberedsaturated, unsaturated, or aromatic heterocycle containing one or moreheteroatoms selected from nitrogen, oxygen, and sulfur, —C(O)O—C₁₋₆allyl, —C(O)O—C₂₋₆ alkenyl, —C(O)O—C₂₋₆ alkynyl, —C(O)O—C₆₋₁₀ saturated,unsaturated, or aromatic carbocycle, —C(O)O-3-12 membered saturated,unsaturated, or aromatic heterocycle containing one or more heteroatomsselected from the group consisting of nitrogen, oxygen, and sulfur, and—C(O)NR₁₃R₁₃, optionally is substituted with one or more R₁₂ groups,

R₁₂ is selected from R₁₄, C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, C₃₋₁₂saturated, unsaturated, or aromatic carbocycle, 3-12 membered saturated,unsaturated, or aromatic heterocycle containing one or more heteroatomsselected from nitrogen, oxygen, and sulfur, optionally substituted withone or more substituents.

Exemplary macrolides are shown in Tables 1 and 2.

TABLE 1 Macrolide Subunits

M1

M2

M3

M4

M5

M6

M7

M8

M9

M10

M11

M12

M13

M14

M15

M16

M17

M18

The pyran-ZBG group can be attached to macrolide at various points asshown in Table 2:

TABLE 2 14-Membered Macrolides

M19

M20

M21

M22

M23

M24

M25

M26

M27

M28

M29

M30

In M25 and M26, the bond between the starred carbon and the nitrogen canbe a single bond or a double bond. This is represented by a dashed linein the structures of M25 and M26.

The zinc binding group may be any moiety or moieties that inhibit(s) theactivity of zinc metalloenzymes such as HDAC. Exemplary zinc bindinggroups include hydroxamates, N-formyl hydroxylamine (orretro-hydroxamate), carboxylates, thiols, dithiols, phosphonic acids,thiadiazoles, sulfodiimines, thiadiazines, trithiocarbonates,thioesters, benzamide, keto groups, mercaptoacetamides, 2-ketoamides,epoxides, epoxyketones, trifluoromethyl ketones, hydroxypyridinones,such as 2-hydroxy and 3-hydroxypyridinones, pyrones,hydroxylpyridinethiones, such as 3-hydroxy-2-methylpyridine-4-thione,and thiopyrones.

In some embodiments, the zinc binding group is a hetercyclic moiety. Inparticular embodiments, the heterocyclic moiety is a heterocyclic groupsubstituted with one or more thiones, hydroxyl groups, thiols, ketones,or combinations thereof. Exemplary zinc binding groups of this typeinclude:

In some embodiments, the zinc binding group can be selected to provideselectivity for a particular HDAC isoform.

In some instances, HDAC inhibitors bearing a zinc binding groupcontaining a heterocyclic group substituted with one or more thionespossesses a higher activity that the same inhibitor bearing a zincbinding group containing a heterocyclic group substituted with one ormore ketones. In certain embodiments, the zinc binding group is a3-hydroxypyridine-2-thione.

The compounds described herein may have one or more chiral centers andthus exist as one or more stereoisomers. Such stereoisomers can exist asa single enantiomer, a mixture of diastereomers or a racemic mixture.

As used herein, the term “stereoisomers” refers to compounds made up ofthe same atoms having the same bond order but having differentthree-dimensional arrangements of atoms which are not interchangeable.The three-dimensional structures are called configurations. As usedherein, the term “enantiomers” refers to two stereoisomers which arenon-superimposable mirror images of one another. As used herein, theterm “optical isomer” is equivalent to the term “enantiomer”. As usedherein the term “diastereomer” refers to two stereoisomers which are notmirror images but also not superimposable. The terms “racemate”,“racemic mixture” or “racemic modification” refer to a mixture of equalparts of enantiomers. The term “chiral center” refers to a carbon atomto which four different groups are attached. Choice of the appropriatechiral column, eluent, and conditions necessary to effect separation ofthe pair of enantiomers is well known to one of ordinary skill in theart using standard techniques (see e.g. Jacques, J. et al.,“Enantiomers, Racemates, and Resolutions”, John Wiley and Sons, Inc.1981).

In one embodiment, the HDAC inhibitors have the formula shown below:

wherein R is:

and m is an integer from 1-12, preferably from 1-10, more preferablyfrom 4-10, most preferably from 5-9 inclusive. However, compounds wherem is greater than 12 may be prepared and their activity determined asdescribed in the examples.

R can be positioned ortho, meta, or para to the triazolyl group. In oneembodiment, R is para to the triazolyl group. In another embodiment, Ris meta to the triazolyl group.

The macrolide can be any suitable maroclide. In one embodiment, themacrolide is selected from M9 or M27-M30 in Table 2.

In another embodiment, the HDAC inhibitors have the following formula:

wherein R and M are as defined above and z is oxygen or —NCO—.

In still another embodiment, the HDAC inhibitor has one of the formulasabove, wherein the zinc binding group is a pyridine thione, diaminobenzene, or hydroxypyridone group.

In another embodiment, the HDAC inhibitors have the formula shown below:

wherein R is:

and m is an integer from 1-12, preferably from 1-10, more preferablyfrom 4-10, most preferably from 5-9 inclusive. However, compounds wherem is greater than 12 may be prepared and their activity determined asdescribed in the examples.

R can be positioned ortho, meta, or para to the triazolyl group. In oneembodiment, R is para to the triazolyl group. In another embodiment, Ris meta to the triazolyl group.

The macrolide can be any suitable maroclide. In one embodiment, themacrolide is selected from M9 or M27-M30 in Table 2.

Non-limiting examples of HDAC inhibitors of Formula I and II are shownin Table 3.

TABLE 3 Non-peptide HDAC Inhibitors COMPOUND NUMBER STRUCTURE  7

 8

 9

 10

 11

 12

 13

 14

 23

 24

 25

 26

 27

 28

 29

 30

 36

 38

 40

 44

 47

 48

 49

 50

 51

 52

 53

 54

 55

 56

 82

 83

 84

 85

 86

 87

 88

 89

 90

 91

 92

 93

 94

 95

 96

 97

 98

 99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

130

131

132

133

134

136

137

It is believed that substitution of the cyclic peptide moiety of aprototypical cyclic-peptide HDAC inhibitor with macrolide skeletons willgenerate a new class of potent HDAC inhibitors. Furthermore, this classof HDAC inhibitors may possess targeted activity, such as targetedanti-cancer activity due to selective tissue distribution conferred bythe macrolide moiety. The biological effects of macrolides are aided bytheir high distribution into target tissues. Macrolides accumulate inhigher concentration within leukocytes as compared to levels found inserum.

III. Formulations

The compounds described herein can be formulated for enteral,parenteral, and/or topical (e.g., transdermal, mucosal, etc.)administration. The compounds and their pharmaceutically-acceptableaddition salts, prodrugs, and/or solvates can also be used in the formof pharmaceutical preparations which facilitate bioavailability. One ormore compounds described herein may be administered in a single dosageform or in multiple dosage forms

Formulations containing one or more of the compounds described hereinmay be prepared using a pharmaceutically acceptable carrier composed ofmaterials that are considered safe and effective and may be administeredto an individual without causing undesirable biological side effects orunwanted interactions. The carrier is all components present in thepharmaceutical formulation other than the active ingredient oringredients. As generally used herein “carrier” includes, but is notlimited to, diluents, binders, lubricants, disintegrators, fillers, pHmodifying agents, preservatives, antioxidants, solubility enhancers, andcoating compositions.

Carrier also includes all components of coating compositions which mayinclude plasticizers, pigments, colorants, stabilizing agents, andglidants. Delayed release, extended release, and/or pulsatile releasedosage formulations may be prepared as described in standard referencessuch as “Pharmaceutical dosage form tablets”, eds. Liberman et. al. (NewYork, Marcel Dekker, Inc., 1989), “Remington—The science and practice ofpharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, Md.,2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6thEdition, Ansel et al., (Media, Pa.: Williams and Wilkins, 1995). Thesereferences provide information on carriers, materials, equipment andprocess for preparing tablets and capsules and delayed release dosageforms of tablets, capsules, and granules.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients present in thedrug-containing tablets, beads, granules or particles include, but arenot limited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants. Diluents, also referred to as “fillers,”are typically necessary to increase the bulk of a solid dosage form sothat a practical size is provided for compression of tablets orformation of beads and granules. Suitable diluents include, but are notlimited to, dicalcium phosphate dihydrate, calcium sulfate, lactose,sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose,kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinizedstarch, silicone dioxide, titanium oxide, magnesium aluminum silicateand powdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard drug decomposition reactionswhich include, by way of example, oxidative reactions.

Surfactants may be anionic, cationic, amphoteric or nonionic surfaceactive agents. Suitable anionic surfactants include, but are not limitedto, those containing carboxylate, sulfonate and sulfate ions. Examplesof anionic surfactants include sodium, potassium, ammonium of long chainalkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine,

If desired, the tablets, beads, granules, or particles may also containminor amount of nontoxic auxiliary substances such as wetting oremulsifying agents, dyes, pH buffering agents, or preservatives.

Enteral Formulations

Pharmaceutical compositions for oral administration can be liquid orsolid. Liquid dosage forms suitable for oral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto an encapsulated or unencapsulated HDAC inhibitor, the liquid dosageforms may contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurylalcohol, polyethylene glycols and fatty acid esters of sorbitan andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants, wetting agents, emulsifying and suspending agents,sweetening, flavoring and perfuming agents.

Solid dosage forms for oral administration include, but are not limitedto, capsules, tablets, caplets, dragees, powders and granules. In suchsolid dosage forms, the encapsulated or unencapsulated compound istypically mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or (a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol and silicic acid, (b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose and acacia, (c) humectants such as glycerol, (d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates and sodium carbonate, (e) solutionretarding agents such as paraffin, (f) absorption accelerators such asquaternary ammonium compounds, (g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolinand bentonite clay and (i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfateand mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also contain buffering agents.

Solid compositions of a similar type may also be employed as fillmaterials in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugar as well as high molecular weight polyethyleneglycols and the like. The solid dosage forms of tablets, dragees,capsules, pills and granules can be prepared with coatings and shellssuch as enteric coatings and other coatings well known in thepharmaceutical formulating art.

Parenteral Formulations

Pharmaceutical preparations in the form suitable for injection aresubjected to conventional pharmaceutical operations such assterilization and/or may contain adjuvants including, but not limitedto, preservatives, stabilizers, wetting or emulsifying agents, andbuffers.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated as known in the art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution,suspension, or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils can be employed as a solvent or suspendingmedium. For this purpose any bland fixed oil can be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid can be used in the preparation of injectable formulations. In aparticularly preferred embodiment, the compound is suspended in acarrier fluid containing 1% (w/v) sodium carboxymethyl cellulose and0.1% (v/v) TWEEN™ 80. The injectable formulations can be sterilized, forexample, by filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium prior to use.

Topical Formulations.

The compounds described here can also be formulated for topical,transdermal, or mucosal delivery. Dosage forms for topical ortransdermal administration include, but are not limited to, ointments,pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, orpatches. The compounds are typically admixed under sterile conditionswith a pharmaceutically acceptable carrier and any excipients (e.g.,preservatives, buffers, etc.) that may be required. Ophthalmicformulations, ear drops and eye drops can also be prepared. Theointments, pastes, creams and gels may contain, in addition to theactive agent, excipients such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc and zinc oxide, ormixtures thereof.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compounds described herein in a propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the compound(s)in a polymer matrix or gel.

Powders and sprays can contain, in addition to the active agent,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these drugs.Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds describedherein with suitable non-irritating excipients or carriers such as cocoabutter, polyethylene glycol, or a suppository wax which are solid atambient temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the compound(s).

A. Other Active Agents

The HDAC inhibitors described herein can be administered adjunctivelywith other active compounds. These compounds include but are not limitedto analgesics, anti-inflammatory drugs, antipyretics, antidepressants,antiepileptics, antihistamines, antimigraine drugs, antimuscarinics,anxioltyics, sedatives, hypnotics, antipsychotics, bronchodilators,anti-asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics,electrolytes, gastro-intestinal drugs, muscle relaxants, nutritionalagents, vitamins, parasympathomimetics, stimulants, anorectics andanti-narcoleptics. “Adjunctive administration”, as used herein, meansthe HDAC inhibitors can be administered in the same dosage form or inseparate dosage forms with one or more other active agents.

Specific examples of compounds that can be adjunctively administeredwith the GDAC inhibitors include, but are not limited to, aceclofenac,acetaminophen, adomexetine, almotriptan, alprazolam, amantadine,amcinonide, aminocyclopropane, amitriptyline, amolodipine, amoxapine,amphetamine, aripiprazole, aspirin, atomoxetine, azasetron, azatadine,beclomethasone, benactyzine, benoxaprofen, bermoprofen, betamethasone,bicifadine, bromocriptine, budesonide, buprenorphine, bupropion,buspirone, butorphanol, butriptyline, caffeine, carbamazepine,carbidopa, carisoprodol, celecoxib, chlordiazepoxide, chlorpromazine,choline salicylate, citalopram, clomipramine, clonazepam, clonidine,clonitazene, clorazepate, clotiazepam, cloxazolam, clozapine, codeine,corticosterone, cortisone, cyclobenzaprine, cyproheptadine,demexiptiline, desipramine, desomorphine, dexamethasone, dexanabinol,dextroamphetamine sulfate, dextromoramide, dextropropoxyphene, dezocine,diazepam, dibenzepin, diclofenac sodium, diflunisal, dihydrocodeine,dihydroergotamine, dihydromorphine, dimetacrine, divalproxex,dizatriptan, dolasetron, donepezil, dothiepin, doxepin, duloxetine,ergotamine, escitalopram, estazolam, ethosuximide, etodolac, femoxetine,fenamates, fenoprofen, fentanyl, fludiazepam, fluoxetine, fluphenazine,flurazepam, flurbiprofen, flutazolam, fluvoxamine, frovatriptan,gabapentin, galantamine, gepirone, ginko bilboa, granisetron,haloperidol, huperzine A, hydrocodone, hydrocortisone, hydromorphone,hydroxyzine, ibuprofen, imipramine, indiplon, indomethacin, indoprofen,iprindole, ipsapirone, ketaserin, ketoprofen, ketorolac, lesopitron,levodopa, lipase, lofepramine, lorazepam, loxapine, maprotiline,mazindol, mefenamic acid, melatonin, melitracen, memantine, meperidine,meprobamate, mesalamine, metapramine, metaxalone, methadone, methadone,methamphetamine, methocarbamol, methyldopa, methylphenidate,methylsalicylate, methysergid(e), metoclopramide, mianserin,mifepristone, milnacipran, minaprine, mirtazapine, moclobemide,modafinil (an anti-narcoleptic), molindone, morphine, morphinehydrochloride, nabumetone, nadolol, naproxen, naratriptan, nefazodone,neurontin, nomifensine, nortriptyline, olanzapine, olsalazine,ondansetron, opipramol, orphenadrine, oxaflozane, oxaprazin, oxazepam,oxitriptan, oxycodone, oxymorphone, pancrelipase, parecoxib, paroxetine,pemoline, pentazocine, pepsin, perphenazine, phenacetin,phendimetrazine, phemnetrazine, phenylbutazone, phenytoin,phosphatidylserine, pimozide, pirlindole, piroxicam, pizotifen,pizotyline, pramipexole, prednisolone, prednisone, pregabalin,propanolol, propizepine, propoxyphene, protriptyline, quazepam,quinupramine, reboxitine, reserpine, risperidone, ritanserin,rivastigmine, rizatriptan, rofecoxib, ropinirole, rotigotine, salsalate,sertraline, sibutramine, sildenafil, sulfasalazine, sulindac,sumatriptan, tacrine, temazepam, tetrabenozine, thiazides, thioridazine,thiothixene, tiapride, tiasipirone, tizanidine, tofenacin, tolmetin,toloxatone, topiramate, tramadol, trazodone, triazolam, trifluoperazine,trimethobenzamide, trimipramine, tropisetron, valdecoxib, valproic acid,venlafaxine, viloxazine, vitamin E, zimeldine, ziprasidone,zolmitriptan, zolpidem, zopiclone and isomers, salts, and combinationsthereof.

IV. Methods of Preparation

Compound numberings, e.g., 1, 2, 3, etc. as used below are for referencewithin this section only are not to be confused with any similarnumberings in the synthetic schemes described below or in the examples.

The schemes below describe exemplary chemistries that can be used tosynthesize the compounds described herein. It is to be appreciated thecompounds described herein may be synthesized by other methods known inthe art.

General Synthetic Schemes

Scheme 1a-c illustrates representative general syntheses of compounds oftypes 5 and 6, 9 and 10, and 12 and 13. The starting des-N-methylmacrolide 1 could be sourced from a variety of N-demethylation reactionsof the tertiary amities of basic sugars on macrolides known in the art(see Flynn et al. (1954) J. Am. Chem. Soc., 76: 3121; U.S. Pat. No.3,725,385; Ku et al. (1997) Bioorg. Med. Chem. Lett., 7: 1203; Stenmarket al. (2000) J. Org. Chem., 65: 3875; Randolph et al. (2004) J. Med.Chem., 47, 1085; and U.S. Pat. No. 7,335,753).

Reaction of 1 with electrophiles 2 and 7 yields alkynes and nitriles 3and 8 respectively. Reactions of azide 4 and nitrile oxide 11 withalkynes 3 generate two regioisomeric triazole and isoxazole products 5and 6 and 12 and 13 respectively. The triazole products' regioisomericratios and reaction rates could be altered by heating the reactionand/or by the use of catalysts (such as, but not limited to, copper (I)and Ru (H) salts and complexes: see Rostovtsev et al. (2002) Angew.Chem. Int. Ed., 41: 2596; Tornoe et al. (2002) J. Org. Chem., 67: 3057;Zhang et al. (2005) J. Am. Chem. Soc., 127: 15998). Similarly, reactionof nitrile 8 and azide 4 generates two regioisomeric tetrazole products,9 and 10. The ZBG in azide 4 can be protected if necessary. Suitable ZBGprotecting groups include are described herein.

Scheme 2 illustrates representative general syntheses of compounds withamide as the linker-cap group connection moiety and benzamide compoundsof types 17 and 19 and 21 and 23 respectively. Reaction of 1 withelectrophile 14 yields ester 15. Compound 15 can be directly reactedwith ZBG amine 18 to yield compound 19. Alternatively, 19 can beobtained from acid 16 and ZBG amine 18 through carbodiimide coupling.Additionally, hydroxamate 17, containing an appropriate aromatic moietyand appropriate alkyl chain length can be obtained from the reaction ofhydroxylamine with ester 15. Similarly, benzamide compounds 21 and 23can be prepared from the reaction of ester 15 or acid 16 with anilines20 and 22. In the case of the para-substituted benzamide 23, theintermediate nitro anilide must be reduced to obtain the desiredbenzamide 23. The synthesis of para-substituted nitro aniline 22 isknown in the art (for example, see Moradei et al. (2007) J. Med. Chem.,45, 5543).

Scheme 3 illustrates representative general syntheses of ketolide andbridged-ketolide based triazole derivatives. Clarithromycin 24 can beN-demethylated to give des-N-methyl clarithromycin 25. Reaction of 25with electrophile 2 yields alkyne 26. Alternatively, alkyne 26 can beobtained by reductive amination of appropriate aldehydes and ketones.Descladinose 27 can be prepared from the treatment of alkyne 26 withdilute mineral acid, such as HCl. Compound 27, where R₁₃ is CH₃, can besimilarly obtained from reaction of 24 with dilute mineral acid.Selective acylation of the hydroxyl group of the amine sugar can beachieved by treatment of compound 27 with acetic anhydride inappropriate non-protic solvents such as, but not limited to, acetone, inthe absence of base to yield compound 28. Oxidation of 28 underCorey-Kim or similar conditions (see Corey & Kim (1972) J. Am. Chem.Soc., 94: 7586; Pfitzner & Moffatt (1965) J. Am. Chem. Soc., 87: 5661;Ley et al. (1994) Synthesis, 639) leads to ketolide 29. Treatment of 29with carbonyldiimidazole and NaHMDS will give carbamate 30. Reaction of30 with amines 31, 32 and ethane-1,2-diamine will afford intermediate33, 34, and 35, respectively (see U.S. Pat. No. 5,631,355; Kashimura etal. (2003), J. Antibiot., 56: 1062; Randolph et al. (2004) J. Med.Chem., 47, 1085; Plata et al. (2004) Tetr., 60: 10171). Subsequentreactions of intermediates 33, 34, and 35 with azide 4 will lead tobridged-ketolides 36 and 37, 38 and 39, and 40 and 41, respectively.When R₁₃ contains an appropriate aromatic moiety and alkyl chain length,ketolide 29 can be modified to form compound 30 by methanolysis atelevated temperatures. Subsequent reaction of 30 with azide 4 willfurnish ketolide 43. Again, it should be appreciated that the ZBG inazides of type 4 can be appropriately protected. Moreover, similarchemistries can be applied for the synthesis of the tetrazoles andoxazoles analogs of the ketolide and bridged-ketolide exemplified inscheme 3.

Scheme 4 illustrates representative general synthesis of triazolecompounds with HDAC recognition cap-group connected to the macrocyclicring at O6 position of 14-membered macrolide such as, but not limitingto, erythromycin. Adapting known protocols (see, Plata et al. (2004)Tetra., 60: 10171), the aryl alkyne 47 can be obtained from readilyavailable erythromycin A-9-oxime (see, Morimoto et al. (1990) J.Antibiot., 43:286) through the intermediacy of 45. Sequential basetreatment with aqueous alkali and potassium carbonate in methanol willlead to alkyne 48 which can be reacted with azide 4 to give triazole 49.Methanolysis of 49 will afford triazole 50. Triazole 50 can be modifiedto form compound 51 by treatment with dilute mineral acid.Alternatively, methanolysis of alkyne 48 yields alkyne 51. Reaction of51 with azide 4 will yield triazole 50.

Scheme 5 illustrates representative general syntheses of triazolecompounds with HDAC recognition cap-groups connected to the macrocyclicring at the O6 position of 14-membered ketolides and carbamate modifiedmacrolides. Selective benzoyl deprotection and silyl group removal willafford aryl alkyne 53. The oxime group in 53 can be removed by heating53 in a THF/H₂O containing NaHSO₃ and L-tartaric acid to afford arylalkyne 54 (adapting protocols described by Plata et al. (2004) Tetra.,60: 10171). Reaction of 54 with azide 4 followed by debenzoylation bysequential treatment with potassium carbonate in methanol andmethanolysis at elevated temperatures will yield triazole 55. Triazole55 can be modified to form compound 56 by treatment with dilute mineralacid. Aryl alkyne 54 can be modified to form aryl alkyne 57 by adaptingprocedures exemplified for similar transformations in scheme 3 oralternative protocols described in the art (see U.S. Pat. No. 5,631,355;Kashimura et al. (2003), J. Antibiot., 56: 1062; Randolph et al. (2004)J. Med. Chem., 47, 1085; Plata et al. (2004) Tetra., 60: 10171).Reaction of 57 with azide 4 followed by debenzoylation by sequentialtreatment with potassium carbonate in methanol and methanolysis atelevated temperatures will yield triazole 58. Triazole 58 can beconverted to compound 59 by treatment with dilute mineral acid. A directtreatment of 57 with dilute mineral acid will afford alcohol 60.Oxidation of 60 under Corey-Kim or similar conditions followed bymethanolysis at elevated temperatures will yield alkyne ketolide 61.Reaction of 61 with azide 4 will yield triazole 62.

Intermediates such as electrophiles 2, 7 and 14, azide 4, nitrile oxide11, amine 30 and carbonate alkyne 46 are all easily accessible bysynthetic protocols known in the art. Exemplary examples for thesynthesis of compounds whose general structures fit the forgoing areillustrated in schemes 6, 7, 8, and 9. Halogenated aryl alkyne 63 canundergo Heck coupling with ethyl acrylate to furnish α,β-unsaturatedester 64. Reduction of 64 with DIBAL should generate alkenol 65, whichcan be transformed to carbonate 66 using methods known in the art (seeU.S. Pat. No. 6,579,986; Plata et al. (2004), Tetra., 60: 10171).Alternatively, alkenol 65 can be prepared from alkenol 67 throughHagihara-Sonogashira coupling (Belema et al. Tet. Lett., (2004), 45:1693). Reactions of carboxylic acid 68 with thionyl chloride in methanolfollow by treatment with sodium azide should furnish azido methyl ester69. Treatment of 69 with hydroxylamine should provide azido hydroxamate70 (Ho et al., (2005), J. Org, Chem., 70, 4873). The hydroxamate groupof 70 can be appropriately protected with a silyl group to provide silylazide 71 (Muri et al. Org. Lett., (2000) 2: 539). Hagihara-Sonogashiracoupling between alcohol 72 and TMS-acetylene should yield alkyne 73.TMS deprotection should furnish alkyne 74, which can be reacted withMsCl to provide mesylate 75. Reaction of phthalimide salt with 75 shouldprovide phthalimide 76, which can be converted to amine 77 byhydrazinolysis or other suitable protocol known in the art. Treatment ofalcohol 78, incorporating ZBG (appropriately protected when necessary),with oxidants such as PDC, should provide aldehyde 79. Reaction of 79with hydroxylamine should furnish oxime 80, which can be converted tonitrile oxide 81 by treatment with NBS or other reagents such NCS,chloramine T, etc. Because of the likely instability of nitrile oxides,the generation of nitrile oxide can be performed in the presence of theappropriate alkyne 3 (scheme 1) to furnish the regioisomeric mixture ofthe isoxazoles.

Schemes 10 and 11 illustrate the synthesis of compounds 7-14 and 23-30in Table 3.

Scheme 12 illustrates the synthesis of compounds 36 and 38 in Table 3.

Schemes 13, 14 and 15 below illustrate the synthesis of compounds 40, 44and 47 in Table 3.

Schemes 16A and 16B. Synthesis of Compound 56 Compound 56 can besynthesized via the 4′-ethynylbenzylketolide intermediate 9.Demethylation of clarithromycin 1 under standard conditions forms4-desmethyl-clarithromycin 2 in 70% yield. Subsequent alkylation of 2with 4-ethynylbenzyl methanesulfonate afforded the modified4′-ethynylbenzylclarithromycin 4 which was treated with 1N HCl toselectively cleave the cladinose sugar to form compound 5. Reaction timefor this step should be carefully controlled as longer reaction timesled to the formation of significant amounts of byproduct. Selectiveacetylation of the 2′-OH group was accomplished by treating an acetonesolution of 5 with acetic anhydride at 40° C. for 24 h to give compound6 in 70% yield. Subsequent oxidation of the 3-hydroxyl group of compound6 to a 3-keto functional group, under anhydrous condition with NCS,afforded the ketolide 7 almost quantitatively. Treatment of 7 withexcess carbonyldiimidazole (CDI) and NaHMDS in a mixture of THF/DMFafforded the 12-acyl imidazolide 8 in 52% yield. Transformation into thedesired tricyclic ketolide was achieved in two successive cyclizationsteps adapting literature procedures. Reaction of imidazolide 8 withethylenediamine followed by intramolecular Michael addition led to theformation of 11,12-cyclic carbamate. Subsequent intramoleculardehydration completed the cyclization process, affording the desiredproduct 9 in 42% yield (Scheme 16A).

Copper (I) catalyzed cycloaddition of 9 with O-trityl protectedazidohydoxamate analogs 10a-e (route A), afforded the 1,2,3-triazolelinked derivatives 11a-e. Deprotection of the trityl group in 11a-e withTFA (route 13) gave the desired products (56a-e) in good yields. Asimilar outcome was obtained when trityl group deprotection was effectedwith BF₃.OEt₂ (route C). Additionally, the desired hydroxamates could beobtained through direct copper (I) catalyzed cycloaddition betweenunprotected azidohydroxamate 13 and alkyne 9 (route D).

Methods of Purifying HDAC Intermediates on Multi-Gram Scales

To improve the efficiency of the multi-gram synthesis, methods ofpurifying intermediates, such as compound B below (where R is amacrolide such as azithromycin or clarithromycin) by extraction weredeveloped.

Compound B containing an azithromycin macrolide can be purified bydissolving B in a suitable alcohol, such as methanol, and adding anaqueous buffer solution, such as an acetate buffer, to form a weaklyacidic solution. The solution can then be extracted with one or moremixtures of a polar and non-polar organic solvent, such as mixtures ofhexanes and ethyl acetate. The aqueous phase is then basified, forexample by addition of aqueous ammonium hydroxide, and extracted withone or more organic solvents, such as hexanes and/or mixtures of hexanesand ethyl acetate. The organic phases can then be collected and dried invacu to obtain B.

Compound B containing a clarithromycin macrolide can be purified bydissolving B in a suitable alcohol, such as methanol, and adding anaqueous buffer solution, such as an acetate buffer, to four a weaklyacidic solution. The solution can then be extracted with one or moremixtures of a polar and non-polar organic solvent, such as mixtures ofhexanes and ethyl acetate. The organic phases can then be collected anddried in vacu. Compound B containing a clarithromycin macrolide can bepurified by recrystallization, for example, from a mixture of hexanesand dichloromethane.

V. Methods of Use

The compounds described herein may be used as anti-cancer agents,anti-inflammatory agents, anti-infective agents, anti-malarial agents,cytoprotective agents, neuroprotective agents, chemopreventive agents,prokinetic agents, and/or cognitive enhancing agents. Examples of cancerwhich may be treated include, but are not limited to, epithelialcancers, such as melanoma, lung cancer, breast cancer, pancreaticcancer, ovarian cancer, prostate cancer, colon cancer, and bladdercancer; hematological cancers, such as multiple myeloma, leukemia, andlymphoma; cervical cancer; and liver cancer.

The potency of the compounds described herein were investigated bydetermining the drug concentrations necessary for 50% inhibition of cellviability (IC₅₀) in SKMES 1, NCI-H69, DU 145 cells, lung fibroblasts,and HMEC. Drug concentrations necessary for 50% inhibition of cellviability (EC₅₀) were quantitatively measured using trypan blueexclusion according to literature protocol. Table 5 in the examplesshows the EC₅₀ values for each compound. All compounds inhibited theproliferation of the transformed cells studied with EC₅₀ in the lowmicromolar range. Most importantly, the compounds we less toxic tountransformed cell-lines (lung fibroblast and HMEC).

The compounds described herein were also evaluated for parasiticactivity against Plasmodium falciparum and Leismania donovani. P.falciparum and L. donovani are the causative parasites of malaria andleishmaniasis, two human diseases which constitute a serious threat topublic health in tropical and sub-tropical countries. Antimalarialactivity was evaluated in vitro using chloroquine-sensitive (D6, SierraLeone) and chloroquine-resistant (W2, Indochina) strains of P.falciparum. Antileishmanial activity was evaluated in vitro against thepromastigote stage of L. donovani.

Plasmodium growth inhibition was determined by a parasite lactatedehydrogenase assay using Malstat reagent. Inhibition of viability ofthe promastigote stage of L. donovani was determined using standardAlamar blue assay, modified to a fluorometric assay. Amphotericin B andpentamidine, standard antileishmanial agents; chloroquine andartemisinin, standard antimalarials; and suberoylanilide hydroxamic acid(SAHA), a standard HDAC inhibitor (HDACi) were all used as positivecontrols. To determine selective toxicity index, all compounds weretested against nontransformed mammalian cell lines namely, monkey kidneyfibroblasts (Vero) and murine macrophages (J774.1) using Neutral Redassay.

The non-peptide macrocyclic HDAC inhibitors potently inhibited theproliferation of both the sensitive and resistant strains of P.falciparum with an IC₅₀ ranging from 0.1 μg/mL to 3.5 μg/mL. Inparticular, compounds 5-8 in Table 16, derived from either the 14- or15-membered macrolide analogs and having 6 methylene spacers separatingthe triazole ring from the zinc-binding hydroxamic acid group (n=6), hadthe most potent antimalarial activities in this series. These compoundsare equipotent or >4-fold more potent than the control compound SAHA.Moreover, they are several folds more selectively toxic to eitherstrains of P. falciparum compared to SAHA.

The antimalarial activities of these macrocyclic HDACi followed asimilar trend as their anti-HDAC activity against HDAC1/2 from HeLanuclear extract, suggesting that parasite HDACs could be anintracellular target of the these compounds.

Compounds 5 and 9 exhibited modest activity against the promastigotestage of L. donovani. A comparison of the antileishmanial activities ofcompounds 13 and 14, analogs with n=8, revealed a disparity between theactivity of 14- and 15-membered macrocyclic rings. 14-Membered compound13 is 5- to 6-fold more potent than its 15-membered congener 14.However, this disparity dissipates after a single methylene groupextension (n=9), as compounds 15 and 16 have virtually indistinguishableantileishmanial activities. Comparatively, compounds 13, 15 and 16,analogs with the most potent antileishmanial activities, are about 7- to10-fold more potent than SAHA and approximately 3-fold less potent thanpentamidine.

Since these non-peptide macrocyclic HDACi have nanomolar anti-HDACactivities, the observed disparity in the trend of their antimalarialand antileishmanial activities may have implication in the organizationof the active sites of the relevant P. falciparum and L. donovani HDACisozymes. These observations provide additional evidence of thesuitability of HDAC inhibition as a viable therapeutic option to curbinfections caused by apicomplexan protozoans and trypanosomatids (1, 5,6, 14) and could facilitate the identification of other HDACi that aremore selective for either parasite.

The formulations contain an effective amount of one or more HDACinhibitors. The doses in which the HDAC inhibitors and their salts,prodrugs, or solvates can be administered may vary widely depending onthe condition of the patient and the symptoms to be treated. One ofordinary skill in the art can readily determine the necessary dosagebased on the condition of the patient and the disease to be treated.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

EXAMPLES Materials

“Preparative TLC” or “prep TLC” refers to preparative thin layerchromatography and was performed on Analtech preparative TLC plates (UV254, 2000 μm), unless otherwise stated. “Column chromatography” or“flash column chromatography” was performed with 200-400 Mesh silicagel, unless otherwise noted.

Nuclear magnetic resonance (NMR) spectra were recorded on aVarian-Gemini 400 magnetic resonance spectrometer. ¹H NMR spectra wererecorded in parts per million (ppm) relative to the peak of CDCl₃, (7.24ppm), CD₃OD (3.31 ppm), or DMSO-d₆ (2.49 ppm). ¹³C spectra were recordedrelative to the central peak of the CDCl₃ triplet (77.0 ppm), CD₃OD(49.0 ppm), or the DMSO-d₆ septet (39.7 ppm), and were recorded withcomplete hetero-decoupling.

Common reaction solvents were either high performance liquidchromatography (HPLC) grade or American Chemical Society (ACS) grade,and used without further purification. Anhydrous solvents and otherreagents were purchased and used without further purification.

Fluor de Lys™ is a fluorescence based HDAC activity assay comprising acombination of fluorogenic Histone deAcetylase Lysyl substrate and adeveloper. The kit is a highly sensitive and convenient alternative toradiolabeled, acetylated histones or peptide/HPLC methods for the assayof histone deacetylases. This assay is based on the ability of HeLanuclear extract, which is enriched in HDAC activity, to mediate thedeacetylation of the acetylated lysine side chain of the Fluor de Lyssubstrate. The assay procedure requires two steps. First, incubation ofthe HeLa nuclear extract with the Fluor de Lys substrate results insubstrate deacetylation and thus sensitizes it to the second step. Inthe second step, treatment of the deacetylated substrate with the Fluorde Lys developer produces a fluorophore. The substrate-developerreaction, under normal circumstances goes to completion in less than 1min at 25° C. The kit used was the Fluorimetric Assay/Drug DiscoveryKit—AK-500 Manual Fluorescent Assay System available from BIOMOL®International, Plymouth Meeting, Pa.

The numbers used to identify the compounds described in the examplescorrespond to the references numbers in Table 3 and/or reaction schemes10-15.

Example 1 Synthesis of Compounds 7-14 and 23-30 in Table 3 Synthesis ofAzithromycin-N-phenylacetylene (3)

To a solution of N-demethylated azithromycin 1 (2.0 g, 2.56 mmol) inanhydrous DMSO (30 ml) was added Hunig's base (4 ml) and 4-ethynylbenzylmethanesulfonate 2 (0.760 g, 3.60 mmol). The reaction mixture was heatedwith stirring under argon at 85° C. for 2.5 h. The reaction was cooledand diluted with ethyl acetate (EtOAc, 100 mL) and washed with saturatedNaHCO₃ (3×60 mL) and saturated brine (60 mL). The organic layer wasdried over Na₂SO₄ and concentrated in vacuo. The crude product waspurified by flash chromatography (silica, 12:1:0.05 CH₂Cl₂/MeOH/conc.NH₄OH) to give 1.2 g (52%) of 3 as a brownish white solid. ¹H-NMR(CDCl₃, 400 MHz) δ 0.84 (m), 0.97 (d, J=7.6 Hz), 1.04 (m), 1.12-1.32(m), 1.36-1.53 (m), 1.66-1.75 (m), 1.81-2.07 (m), 2.19 (s), 2.25-2.29(m), 2.48 (m), 2.63-2.73 (m), 2.89 (bs), 2.96 (1, J=9.8 Hz), 3.02 (s),3.08 (s), 3.27-3.32 (m), 3.38-3.45 (m), 3.56 (d, J=6.8 Hz), 3.63 (s),3.72 (d, J=13.2 Hz), 3.97 (m), 4.19 (m), 4.36 (d, J=7.2 Hz), 4.63 (d,J=10 Hz), 5.04 (d, J=4.4 Hz), 7.21 (d, J=8 Hz), 7.39 (d, J=8 Hz);¹³C-NMR (CDCl₃, 100 MHz) δ 7.5, 9.2, 11.3, 14.9, 16.3, 18.3, 21.3, 21.4,21.6, 22.0, 26.8, 27.6, 29.6, 34.7, 36.3, 36.9, 42.0, 42.3, 45.2, 49.2,57.7, 62.3, 63.7, 65.4, 68.5, 70.0, 70.6, 72.7, 73.5, 73.8, 74.2, 77.1,77.9, 78.0, 83.4, 83.7, 94.5, 102.6, 120.8, 128.5, 132.0, 139.6, 178.3;HRMS (FAB, mnba) calc for [C₄₆H₇₆N₂O₁₂+H]⁺ 849.5476. found 849.5411.

Synthesis of Azithromycin-arylalkyltriazolyl methyl ester (17)

Azithromycin-N-phenylacetylene 3 (0.045 g, 0.053 mmol) and azido-ester16 (0.014 g, 0.080 mmol) were dissolved in anhydrous THF (5 mL) andstirred under argon at room temperature. Copper (I) iodide (0.010 g,0.053 mmol), and Hunig's base (0.05 mL) were then added to the reactionmixture, and stirring continued for 12 h. The reaction mixture wasdiluted with CH₂Cl₂ (30 mL) and washed with 1:4 NH₄OH/saturated NH₄Cl(3×25 mL) and again with saturated NH₄Cl (25 mL). The organic layer wasdried over Na₂SO₄ and concentrated under vacuum. The crude product waspurified by preparative TLC, eluting with Hexane/EtOAc/Et₃N 3:2:0.1 togive 50 mg (92%) of 17 as a white-brown solid. ¹H-NMR (CDCl₃, 400 MHz) δ0.82-0.90 (m), 0.98 (d, J=7.6 Hz), 1.05-1.13 (m), 1.19-1.23 (m),1.25-1.30 (m), 1.40-1.52 (m), 1.60-1.74 (m), 1.80-1.96 (m), 2.00-2.06(m), 2.22-2.37 (m), 2.56 (m), 2.67 (m), 2.95 (t, J=9.8 Hz), 3.07 (s),3.29-3.34 (m), 3.46 (bs), 3.54 (d, J=6.8 Hz), 3.61 (s), 3.68 (bs), 3.77(m), 3.97 (m), 4.18 (m), 4.34-4.38 (m), 4.69 (m), 5.06 (d, J=4 Hz), 7.32(d, J=6.4 Hz), 7.73-7.75 (m); ¹³C-NMR (CDCl₃, 100 MHz) δ 8.7, 9.2, 11.3,14.2, 14.7, 16.5, 18.2, 21.4, 21.5, 22.2, 24.2, 25.9, 26.6, 27.3, 29.7,30.0, 33.6, 34.6, 36.4, 36.9, 42.4, 45.3, 45.8, 49.3, 50.0, 51.5, 57.7,63.9, 65.5, 68.6, 69.4, 70.5, 72.7, 73.8, 74.2, 77.2, 77.6, 78.0, 83.4,94.4, 102.7, 119.3, 125.5, 129.1, 129.4, 147.2, 173.4, 178.1. MS (FAB,mnba) 1020.3 (M+H)⁺.

Synthesis of Descladinoseazithromycin-arylalkyltriazolyl methyl ester(18)

A mixture of compound 3 (0.12 g, 0.14 mmol) in 0.25 N HCl (15 mL) wasstirred at room temperature for 20 h and poured into EtOAc (20 mL). Thetwo layers were separated and the aqueous layer was washed with EtOAc(2×20 mL), basified with concentrated NH₄OH and then extracted with 5%MeOH in CH₂Cl₂ (2×30 mL). The combined organic layer was washed withsaturated brine (30 mL) and dried over Na₂SO₄. Solvent was evaporatedoff to give 89 mg (91%) of descladinose compound 4 as a white solid.¹H-NMR (CDCl₃, 400 MHz) δ 0.81-0.87 (m), 0.90-1.07 (m), 1.17-1.27 (m),1.31-1.59 (m), 1.68-1.71 (m), 1.80-1.87 (m), 1.99-2.03 (m), 2.08 (s),2.23-2.27 (m), 2.31 (s), 2.44-2.48 (m), 2.59-2.73 (m), 3.32-3.39 (m),3.48-3.53 (m), 3.60-3.65 (m), 3.73 (d, J=9.6 Hz), 3.84-3.91 (m), 4.43(d, J=7.2 Hz), 4.69-4.72 (m), 7.16 (d, J=8.0 Hz), 7.39 (d, J=8.4 Hz);¹³C NMR (CDCl₃, 100 MHz) δ 7.7, 7.9, 10.9, 14.2, 16.1, 20.9, 21.1, 21.2,25.9, 26.6, 29.3, 36.0, 36.4, 37.1, 42.1, 44.5, 57.6, 60.3, 62.5, 65.3,69.9, 70.5, 70.9, 73.0, 74.1, 75.4, 77.2, 79.5, 83.3, 94.9, 106.5,120.8, 128.3, 132.0, 139.3, 177.2. MS (FAB, mnba) 691.2 (M+H)⁺.

The descladinose compound 4 (0.080 g, 0.115 mmol) and azido-ester 16(0.030 g, 0.173 mmol) were dissolved in anhydrous THF (5 mL) and stirredunder argon at room temperature. Copper (I) iodide (0.010 g, 0.053mmol), and Hunig's base (0.05 mL) were then added to the reactionmixture, and stirring continued for 12 h. The reaction mixture wasdiluted with CH₂Cl₂ (30 mL) and washed with 1:4 NH₄OH/saturated NH₄Cl(3×25 mL) and again with saturated NH₄Cl (25 mL). The organic layer wasdried over Na₂SO₄ and concentrated in vacuo. The crude product waspurified by preparative TLC, eluting with Hexane/EtOAc/Et₃N 3:2:0.1 togive 65 mg (65%) of 18 as a white-brown solid. ¹H-NMR (CDCl₃, 400 MHz) δ0.79-0.86 (m), 1.00-1.07 (m), 1.17-1.35 (m), 1.42-1.51 (m), 1.55-1.72(m), 1.80-1.94 (m), 2.00-2.05 (m), 2.1 (s), 2.23-2.27 (m), 2.33 (s),2.47 (d, J=10.4 Hz), 2.58-2.72 (m), 3.32-3.41 (m), 3.52-3.73 (m),3.92-4.00 (m), 4.34 (t, J=7.0 Hz), 4.41 (d, J=7.6 Hz), 4.69 (d, J=10.8Hz), 7.24 (d, J=8.4 Hz), 7.71 (d, J=8 Hz), 7.73 (s); ¹³C NMR (CDCl₃, 100MHz) δ 7.7, 7.9, 8.7, 10.9, 16.1, 16.1, 20.9, 21.2, 24.2, 25.8, 25.9,26.6, 29.2, 29.6, 30.0, 33.6, 36.0, 36.3, 37.1, 42.0, 44.5, 45.8, 50.0,51.5, 57.7, 62.6, 65.1, 69.9, 70.4, 73.1, 74.1, 75.3, 79.4, 94.8, 106.4,119.3, 125.5, 128.9, 129.6, 138.2, 147.1, 173.4, 177.2. MS (FAB, mnba)862.2 (M+H)⁺.

Synthesis of Azithromycin-N-phenyltriazolylhexahydroxamic acid (7)

Method A

To a solution of compound 17 (0.04 g, 0.04 mmol) in 1:1 THF/MeOH (3 mL)was added hydroxylamine (50% in H₂O) (0.03 mL, 0.54 mmol) and acatalytic amount of KCN. The mixture was stirred at room temperature for24 h. The reaction was partitioned between 5% MeOH in CH₂Cl₂ (30 mL) andsaturated sodium bicarbonate (25 mL), the two layers were separated andthe aqueous layer was extracted with 5% MeOH in CH₂Cl₂ (2×20 mL). Thecombined organic layer was washed with saturated brine (40 mL) and driedover Na₂SO₄. Solvent was evaporated off and the crude was purified bypreparative TLC, eluting with CH₂Cl₂/MeOH/NH4OH 10:1:0.1 to givecompound 7 (6.5 mg, 16%) as brown-white solid.

Method B

Azithromycin-N-phenylacetylene 3 (0.100 g, 0.109 mmol) and6-azidohexahydroxamic acid 15a (0.081 g, 0.117 mmol) were dissolved inanhydrous THF (5 mL) and stirred under argon at room temperature. Copper(I) iodide (0.011 g, 0.07 mmol) and Hunig's base (0.5 mL) were thenadded to the reaction mixture, and stirring continued for 4 h. Thereaction mixture was diluted with CH₂Cl₂ (40 mL) and washed with 1:4NH₄OH/saturated NH₄Cl (3×30 mL) and saturated NH₄Cl (30 mL). The organiclayer was dried over Na₂SO₄ and concentrated in vacuo. The crude productwas purified by prep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/cone. NH₄OH) togive 71 mg (59%) of 7 as a brownish white solid.

Method C

Azithromycin-N-phenylacetylene 3 (0.045 g, 0.050 mmol) and6-azido-O-silyl hexahydroxamate 6 (0.060 g, 0.146 mmol) were dissolvedin anhydrous THF (5 mL) and stirred under argon at room temperature(Note: compound 6 was prepared from the corresponding azido carboxylicacid, t-BuPh₂SiCl and NaH, according to the procedure described by Muriet al. ORG. LETT (2000) 2: 539). Copper (I) iodide (0.010 g, 0.05 mmol),Hunig's base (0.5 mL) andtris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, (TBTA) (0.016 g,0.030 mmol) were then added to the reaction mixture, and stirringcontinued for 2 h (Note: TBTA was synthesized according to Chen et al.Org. Lett., (2004) 6: 2853). The reaction mixture was diluted withCH₂Cl₂ (40 mL) and washed with 1:4 NH₄OH/saturated NH₄Cl (2×30 mL) andsaturated NH₄Cl (30 mL). The organic layer was dried over Na₂SO₄ andconcentrated in vacuo. The crude product was purified by prep TLC(silica, 12:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) to give 38 mg (60%) of silylprotected compound 6 as a brownish white solid. ¹H-NMR (CDCl₃, 400 MHz)δ 0.82-0.92 (m), 1.00-1.14 (m), 1.17-1.27 (m), 1.30-1.32 (m), 1.35-1.59(m), 1.73-1.92 (m), 2.01-2.15 (m), 2.24-2.32 (m), 2.36 (br s), 2.56 (d,J=10.4 Hz), 2.69 (m), 2.98 (d, J=10 Hz), 3.07-3.09 (m), 3.32-3.36 (In),3.42-3.46 (m), 3.55-3.63 (m), 3.66 (s), 3.78 (d, J=13.2 Hz), 4.01 (m),4.15-4.25 (m), 4.40 (d, J=6.8 Hz), 4.63 (d, J=7.2 Hz), 4.69 (s), 5.10(d, J=4.4 Hz), 7.31-7.43 (m), 7.65-7.75 (m).

To a solution of silyl protected compound 6 (0.025 g, 0.02 mmol) in THF(1 mL) was added 1 M TBAF in THF (0.030 mL, 0.030 mmol) and the mixturewas stirred at room temperature for 2 h during which TLC revealed a nearquantitative conversion to a lower Rf product. The reaction waspartitioned between CH₂Cl₂ (30 mL) and saturated NH₄Cl (25 mL), the twolayers were separated and the organic layer dried over Na₂SO₄ andconcentrated in vacuo. The crude product was purified by prep TLC(silica, 12:1:0.1 CH₂Cl₂/MeOH/Et₃N) to give 15 mg (73%) of 7 as abrownish white solid.

¹H-NMR (Acetone-d6, 400 MHz) δ 0.83-0.92 (m), 1.02 (d, J=7.6 Hz),1.08-1.11 (m), 1.14 (d, J=7.6 Hz), 1.18 (d, J=6 Hz), 1.24-1.29 (m),1.33-1.47 (m), 1.54 (dd, J=4.8 Hz, 15.2 Hz), 1.66 (m), 1.80-2.01 (m),2.06-2.12 (m), 2.18-2.24 (m), 2.26 (s), 2.28-2.31 (m), 2.35-2.41 (m),2.51 (d, J=10 Hz), 2.65-2.96 (m), 3.12 (s), 3.22-3.29 (m), 3.41-3.47(m), 3.54-3.69 (m), 3.81 (d, J=13.2 Hz), 4.11 (m), 4.24 (m), 4.45 (t,J=7.0 Hz), 4.50 (d, J=6.8 Hz), 4.75 (d, J=7.2 Hz), 4.97 (d, J=5.2 Hz),7.42 (d, J=8.0 Hz), 7.84 (d, J=8.0 Hz), 8.35 (s). MS (FAB, mnba) 1021.2(M+H)⁺.

Synthesis of Descladinose-Azithromycin-N-phenyltriazolylhexahydroxamicacid (8)

Method A

To a solution of compound 18 (0.04 g, 0.05 mmol) in 1:1 THF/MeOH (3 mL)was added hydroxylamine (50% in H₂O) (0.04 mL, 0.54 mmol) and acatalytic amount of KCN. The mixture was stirred at room temperature for24 h. The reaction was partitioned between 5% MeOH in CH₂Cl₂ (30 mL) andsaturated sodium bicarbonate (25 mL), the two layers were separated andthe aqueous layer was extracted with 5% MeOH in CH₂Cl₂ (2×20 mL). Thecombined organic layer was washed with saturated brine (40 mL) and driedover Na₂SO₄. Solvent was evaporated off and the crude was purified bypreparative TLC, eluting with CH₂Cl₂/MeOH/NH4OH 10:1:0.1 to givecompound 8 (9.0 mg, 23%) as brown-white solid.

Method B

Reaction of descladinose-azithromycin-N-phenylacetylene 4 (0.134 g,0.188 mmol) and 6-azidohexahydroxamic acid 15a (0.130 g, 0.755 mmol)within 8 h (according to the protocols of Method B described for thesynthesis of compound 7 above), followed by prep TLC (silica, 10:1:0.1CH₂Cl₂/MeOH/conc. NH₄OH) gave 73 mg (43%) of 8 as a brownish whitesolid.

¹H-NMR (Acetone-d6, 400 MHz) δ 0.82-0.90 (m), 1.02 (d, J=7.2 Hz), 1.07(s), 1.09 (d, J=6.8 Hz), 1.18-1.23 (m), 1.28 (bs), 1.31-1.39 (m),1.46-1.56 (m), 1.65 (m), 1.81-1.83 (m), 1.87-1.99 (m), 2.05-2.11 (m),2.18-2.21 (m), 2.18-2.21 (m), 2.24 (s), 2.25-2.29 (m), 2.35 (s), 4.47(d, J=9.2 Hz), 2.61-2.67 (m), 2.70-2.77 (m), 3.30-3.34 (m), 3.41 (m),3.52-3.65 (m), 2.81 (d, J=13.2 Hz), 4.44 (t, J=7.0 Hz), 4.59 (d, J=7.6Hz), 4.87 (dd, J=1.8 Hz, 11.0 Hz), 7.43 (d, J=8.4 Hz), 7.83 (d, J=8.4Hz), 8.34 (m); HMRS (ESI) calcd for [C₄₄H₇₄N₆O₁₁+H]⁺863.5488. found863.5528.

Accordingly, compounds 9-14 and 23-30 (in this section) were synthesizedaccording to the protocols of Method B described for the synthesis ofcompound 7 above.

Synthesis of Azithromycin-N-phenyltriazolylheptahydroxamic acid (9)

Reaction of azithromycin-N-phenylacetylene 3 (0.134 g, 0.158 mmol) and7-azidoheptahydroxamic acid 15b (0.125 g, 0.672 mmol) within 4 h,followed by prep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) gave 93mg (56%) of 9 as a brownish white solid. ¹H-NMR (CDCl₃, 400 MHz) δ0.81-1.51 (m), 1.54-1.65 (m), 1.70-2.14 (m), 2.20-2.38 (m), 2.46-2.56(m), 2.60-2.70 (m), 3.00 (s), 3.31 (t, J=8.8 Hz), 3.38-3.54 (m), 3.60(s), 3.78 (d, J=12.8 Hz), 3.98-4.20 (m), 4.36 (d, J=7.2 Hz), 4.49 (d,J=7.2 Hz), 5.11 (d, J=4.0 Hz), 7.32 (d, J=7.6 Hz), 7.73 (s), 7.75 (d,J=7.6 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 6.6, 8.8, 11.5, 14.4, 16.6, 17.7,21.3, 21.6, 21.8, 25.1, 26.0, 26.7, 27.1, 28.2, 29.2, 29.6, 30.0, 33.1,34.5, 35.7, 36.7, 41.8, 42.7, 45.3, 49.3, 50.3, 50.7, 57.9, 62.7, 63.0,65.8, 68.6, 69.4, 70.4, 72.6, 73.2, 73.8, 77.8, 78.1, 78.2, 83.5, 94.4,102.8, 119.3, 125.7, 129.4, 129.7, 138.4, 147.4, 171.3, 178.4; HMRS(ESI) calcd for [C₅₃H₉₀N₆O₁₄+H]⁺ 1035.6587. found 1035.6628.

Synthesis of Descladinose-Azithromycin-N-phenyltriazolylheptahydroxamicacid (10)

Reaction of descladinose-azithromycin-N-phenylacetylene 4 (0.130 g,0.188 mmol) and 7-azidoheptahydroxamic acid 15b (0.130 g, 0.755 mmol)within 8 h, followed by prep TLC (silica, 10:1:0.1 CH₂Cl₂/MeOH/conc.NH₄OH) gave 78 mg (47%) of 10 as a brownish white solid. ¹H-NMR (CDCl₃,400 MHz) δ 0.66-2.32 (m), 2.47 (d, J=10.8 Hz), 2.63-2.70 (m), 3.34-3.51(m), 3.62-3.69 (m), 4.20-4.40 (m), 4.74 (br s), 7.26 (br s), 7.73 (brs); ¹³C NMR (CDCl₃, 100 MHz) δ 7.4, 7.9, 10.7, 16.0, 16.1, 20.9, 21.1,25.0, 25.7, 26.5, 28.0, 28.9, 29.8, 35.8, 36.3, 36.9, 42.0, 44.4, 50.1,57.9, 62.7, 63.9, 69.9, 70.4, 70.8, 73.3, 74.1, 75.2, 79.5, 94.9, 106.6,119.7, 125.7, 129.2, 129.6, 138.4, 147.3, 177.5; HMRS (ESI) calcd for[C₄₅H₇₆N₆O₁₁+H]⁺ 877.5645. found 877.5665.

Synthesis of Azithromycin-N-phenyltriazolyloctahydroxamic acid (11)

Reaction of azithromycin-N-phenylacetylene 3 (0.10 g, 0.120 mmol) and8-azidooctahydroxamic acid 15c (0.047 g, 0.24 mmol) within 2.5 h,followed by prep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) gave 72mg (58%) of 11 as a brownish white solid. ¹H NMR (CDCl₃, 400 MHz) δ 0.85(t, J=4.0 Hz), 0.87-1.22 (m), 1.29 (s), 1.30-2.28 (m), 2.29 (s),2.30-3.00 (m), 3.10 (s), 3.20-3.79 (m), 3.99-4.03 (m), 4.35-4.40 (m),4.65 (d, J=8.0 Hz), 5.11 (d, J=4.8 Hz), 7.34 (d, J=8.0 Hz), 7.72 (s),7.77 (d, J=8.0 Hz); ¹³C NMR (DMSO-d₆, 400 MHz) δ 7.5, 9.8, 11.6, 15.4,18.2, 19.1, 21.5, 22.1, 22.7, 25.6, 26.3, 26.6, 28.7, 29.0, 29.6, 30.2,32.0, 32.8, 35.2, 36.4, 37.2, 42.2, 45.3, 49.2, 50.1, 58.3, 63.2, 65.4,67.7, 70.8, 73.3, 74.2, 77.0, 78.4, 83.4, 102.8, 121.6, 125.6, 129.7,130.0, 135.0, 147.0, 177.8; HRMS (FAB, thioglycerol) calc for[C₅₄H₉₂N₆O₁₄+H]⁺ 1049.6749. found 1049.6648.

Synthesis of Descladinose-Azithromycin-N-phenyltriazolyloctahydroxamicacid (12)

Reaction of azithromycin-N-phenylacetylene 4 (0.10 g, 0.144 mmol) and8-azidooctahydroxamic acid 15c (0.049 g, 0.246 mmol) within 2.5 h,followed by prep TLC (silica, 10:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) gave 94mg (73%) of 12 as a brownish white solid. HRMS (FAB, thioglycerol) calcfor [C₄₆H₇₉N₆O₁₁+H]⁺ 891.5806. found 891.5910.

Synthesis of Azithromycin-N-phenyltriazolylnonahydroxamic acid (13)

Reaction of azithromycin-N-phenylacetylene 3 (0.10 g, 0.120 mmol) and9-azidononahydroxamic acid 15d (0.043 g, 0.20 mmol) within 2.5 h,followed by prep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) gave 64mg (51%) of 13 as a brownish white solid. ¹H NMR (CDCl₃, 400 MHz) δ0.84-1.30 (m), 1.33-2.26 (m), 2.30 (s), 2.38-2.68 (m), 2.99 (s),3.32-3.84 (m), 4.03-4.08 (m), 4.35-4.41 (m), 4.53 (d, J=8.0 Hz), 5.13(d, J=4.0 Hz), 7.35 (d, J=8.0 Hz), 7.75 (s), 7.78 (d, J=8.0 Hz); ¹³C NMR(DMSO-d₆, 400 MHz) δ 6.9, 9.0, 11.6, 14.7, 16.9, 18.0, 21.6, 21.8, 22.1,25.6, 26.4, 26.9, 27.3, 28.8, 29.0, 29.1, 29.5, 29.9, 30.4, 34.8, 36.0,37.0, 42.1, 43.0, 45.6, 49.5, 50.5, 58.1, 63.5, 66.1, 68.8, 70.7, 72.9,74.1, 78.1, 78.3, 78.5, 83.7, 94.4, 94.7, 103.1, 119.6, 126.0, 129.7,130.0, 147.6, 178.7; LRMS (MALDI) calc for [C₅₅H₉₄N₆O₁₄+H]⁺ 1063.6.found 1063.7.

Synthesis of Azithromycin-N-phenyltriazolyldecahydroxamic acid (14)

Reaction of azithromycin-N-phenylacetylene 3 (0.10 g, 0.120 mmol) and10-azido-decahydroxamic acid 15e (0.045 g, 0.20 mmol) within 4.5 h,followed by prep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) gave 70mg (56%) of 14 as a brownish white solid. ¹H NMR (CDCl₃, 400 MHz) δ0.85-1.36 (m), 1.41-2.24 (m), 2.28, 2.36 (s), 2.33-3.10 (m), 3.05 (s),3.23-3.82 (m), 4.06-4.10 (m), 4.36-4.41 (m), 4.49 (d, J=8.0 Hz), 5.15(d, J=4.0 Hz), 7.34 (d, J=8 Hz), 7.75 (s), 7.78 (d, J=8.0 Hz); ¹³C NMR(DMSO-d₆, 400 MHz) δ 6.7, 9.0, 11.7, 14.6, 16.9, 17.9, 21.6, 21.9, 22.0,25.6, 26.4, 26.8, 27.0, 27.3, 28.8, 29.0, 21.9, 29.3, 29.5, 29.9, 30.3,33.6, 34.9, 35.8, 37.0, 42.1, 43.0, 45.6, 49.6, 50.6, 51.6, 58.0, 62.8,63.9, 66.2, 68.9, 69.6, 70.7, 72.9, 73.5, 74.0, 74.1, 78.2, 78.6, 83.6,94.6, 103.0, 119.6, 126.0, 129.6, 129.9, 138.9, 147.6, 178.6; HRMS(MALDI) calc for [C₅₆H₉₆N₆O₁₄+H]⁺ 1077.7057. found 1077.6971.

Synthesis of Clarithromycin-N-phenylacetylene (20)

To a solution of N-demethylated clarithromycin 19 (2.40 g, 3.34 mmol) inanhydrous DMSO (30 ml) was added Hunig's base (3 ml) and 4-ethynylbenzylmethanesulfonate 2 (0.920 g, 4.34 mmol). The reaction mixture was thenheated with stirring under argon at 85° C. for 2.5 h. The reaction wascooled and diluted with EtOAc (100 mL) and washed with saturated NaHCO₃(3×60 mL) and saturated brine (60 mL). The organic layer was dried overNa₂SO₄ and concentrated in vacuo. The crude product was purified byflash chromatography (silica, gradient 12:1; 10:1; 8:1; CH₂Cl₂/acetone)to give 1.8 g (63%) of 20 as a brownish white solid. ¹H-NMR (CDCl₃, 400MHz) δ 0.82 (t, J=7.2 Hz), 1.03-1.28 (m), 1.37 (s), 1.40-1.55 (m),1.65-1.90 (m), 2.03 (d, J=10.0 Hz), 2.22 (s), 2.30 (d, J=15.2 Hz),2.40-2.60 (m), 2.80-2.90 (m), 2.94-3.00 (m), 3.04 (s), 3.09 (s), 3.16(s), 3.24-3.29 (m), 3.38-3.46 (m), 3.59 (d, J=6.8 Hz), 3.70-3.75 (m),3.88-3.95 (m), 4.37 (d, J=7.2 Hz), 4.88 (d, J=4.4 Hz), 5.02 (dd, J=11.6,2.4 Hz), 7.23 (d, J=12.0 Hz), 7.42 (d, J=8.0 Hz); ¹³C NMR (CDCl₃, 100MHz) δ 9.2, 10.7, 12.4, 16.1, 18.1, 18.7, 19.9, 21.1, 21.5, 29.3, 32.4,34.8, 36.9, 37.2, 39.2, 45.0, 45.2, 49.3, 50.6, 53.4, 57.6, 63.3, 65.6,68.5, 69.0, 70.6, 72.5, 74.2, 76.5, 77.8, 78.1, 78.2, 80.8, 95.8, 102.5,120.9, 128.6, 132.0, 133.5, 139.4, 175.4; HRMS (ESI) calc for[C₄₆H₇₃NO₁₃+H]⁺ 848.5155. found 848. 5181.

Synthesis of Descladinose-Clarithromycin-N-phenylacetylene (21)

To a solution of clarithromycin-N-phenylacetylene 20 (0.500 g, mmol) inethanol (20 mL) was added 1N HCl (20 mL), and stirring continued for 22h at room temperature. The reaction mixture was basified withconcentrated NH₄OH to about pH=9. The reaction mixture was diluted withdistilled water (40 mL) and extracted with EtOAc (3×60 mL). The combinedorganic layers were washed with saturated brine (40 mL), dried overNa₂SO₄, and concentrated in wino. The crude product was purified byflash chromatography (silica, 8:1 CH₂Cl₂/acetone) to give 320 mg (79%)of 21 as a brownish white solid. ¹H-NMR (CDCl₃, 400 MHz) δ 0.82 (t,J=7.6 Hz), 1.09-1.28 (m), 1.34 (s), 1.40-1.55 (m), 1.70-1.74 (m),1.87-1.94 (m), 2.08-2.15 (m), 2.54-2.66 (m), 2.94-2.98 (m), 3.05 (s),3.25 (s), 3.31-3.42 (m), 3.48-3.56 (m), 3.66 (d, J=10.0 Hz), 3.82 (s),3.90 (s), 4.35 (d, J=7.6 Hz), 5.14 (dd, J=10.8, 2.0 Hz), 7.18 (d, J=8.0Hz), 7.42 (d, J=8.4 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 8.4, 10.5, 12.7,15.3, 16.3, 17.8, 18.8, 21.4, 29.2, 35.9, 36.6, 37.5, 38.7, 44.5, 45.5,49.6, 57.8, 65.0, 69.7, 70.1, 70.6, 74.1, 77.9, 78.9, 83.3, 88.5, 106.5,121.0, 128.4, 132.1, 139.1, 174.7; HRMS (ESI) calc for [C₃₈H₅₉NO₁₀+H]⁺690.4212. found 690.4259.

Synthesis of Clarithromycin-N-phenyltriazolylhexahydroxamic acid (23)

Reaction of clarithromycin-N-phenylacetylene 20 (0.100 g, 0.120 mmol)and 6-azidohexahydroxamic acid 15a (0.080 g, 0.470 mmol) within 2.5 h,followed by prep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/cone. NH₄OH) gave 70mg (58%) of 23 as a brownish white solid. ¹H-NMR (CDCl₃, 400 MHz) δ 0.81(t, J=7.6 Hz), 1.03-1.52 (m), 1.62-1.92 (m) 2.04-2.29 (m), 2.48-2.60(m), 2.82-2.90 (m), 2.93-2.99 (m), 3.09 (s), 3.19 (s), 3.28-3.33 (m),3.42-3.46 (m), 3.60 (d, J=7.6 Hz), 3.70-3.80 (m), 3.90-3.98 (m),4.37-4.40 (m), 4.87 (d, J=4.8 Hz), 5.03 (dd, J=11.6, 2.4 Hz), 7.34 (d,J=7.6 Hz), 7.77 (d, J=7.6 Hz), 7.82 (s); ¹³C NMR (CDCl₃, 100 MHz) δ 9.1,10.5, 12.2, 15.9, 17.9, 18.5, 19.7, 20.9, 21.2, 21.4, 24.3, 25.5, 29.4,29.6, 34.7, 36.8, 37.1, 39.0, 39.1, 45.0, 45.1, 49.3, 49.9, 50.5, 53.3,57.5, 63.6, 65.5, 68.5, 69.0, 70.7, 72.4, 74.2, 77.8, 78.2, 80.9, 95.9,102.6, 119.8, 125.6, 129.4, 147.4, 175.8; HMRS (ESI) calcd for[C₅₂H₈₅N₅O₁₅+H]⁺ 1020.6114. found 1020.6121.

Synthesis of Descladinose-Clarithromycin-N-phenyltriazolylhexahydroxamicacid (24)

Reaction of descladinose-clarithromycin-N-phenylacetylene 21 (0.075 g,0.109 mmol) and 6-azidohexahydroxamic acid 15a (0.040 g, 0.233 mmol)within 4 h, followed by prep TLC (silica, 10:1:0.1 CH₂Cl₂/MeOH/conc.NH₄OH) gave 47 mg (51%) of 24 as a brownish white solid. ¹H-NMR (CDCl₃,400 MHz) δ 0.79 (t, J=7.2 Hz), 1.08-1.32 (m), 1.39-1.64 (m), 1.71-1.81(m), 1.82-1.96 (m), 2.04-2.18 (m), 2.51-2.70 (m), 2.92-2.98 (m),3.18-3.38 (m), 3.45-3.55 (m), 3.60-3.74 (m), 3.81 (s), 3.90 (s), 4.33(br s), 5.13 (d, J=10.4 Hz), 7.29 (br s), 7.74 (br s); ¹³C NMR (CDCl₃,100 MHz) δ 8.6, 10.7, 12.9, 15.5, 16.4, 18.0, 19.0, 21.5, 21.6, 29.5,29.9, 36.1, 36.7, 37.7, 39.0, 44.7, 45.7, 49.8, 50.3, 58.2, 64.6, 70.0,70.3, 70.9, 74.4, 78.3, 79.1, 88.5, 106.7, 120.3, 126.1, 129.6, 129.9,147.7, 175.4; HMRS (ESI) calcd for [C₄₄H₇₁N₅O₁₂+H]⁺ 862.5172. found862.5155.

Synthesis of Clarithromycin-N-phenyltriazolylheptahydroxamic acid (25)

Reaction of clarithromycin-N-phenylacetylene 20 (0.130 g, 0.153 mmol)and 7-azidoheptahydroxamic acid 15b (0.105 g, 0.565 mmol) within 2.5 h,followed by prep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) gave 105mg (67%) of 25 as yellowish solid. ¹H-NMR (CDCl₃, 400 MHz) δ 0.82 (t,J=8.0 Hz), 1.04-1.52 (m), 1.67-1.92 (m), 2.14-2.29 (m), 2.52-2.60 (m),2.82-2.90 (m), 2.95-3.00 (m), 3.10 (s), 3.16 (s), 3.27-3.32 (m),3.41-3.46 (m), 3.59 (d, J=6.8 Hz), 3.69-3.79 (m), 3.90-3.95 (m),4.34-4.39 (m), 4.87 (d, J=4.4 Hz), 5.02 (d, J=9.2 Hz), 7.33 (d, J=6.4Hz), 7.77 (d, J=8.4 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 9.1, 10.5, 12.2,15.9, 17.9, 18.5, 19.8, 20.9, 21.2, 21.4, 24.7, 25.5, 27.7, 29.8, 34.7,36.8, 37.1, 39.1, 45.0, 45.2, 49.3, 50.0, 50.5, 57.6, 63.6, 65.6, 68.6,69.0, 70.7, 72.4, 74.2, 77.8, 78.3, 80.9, 95.9, 102.7, 119.5, 125.7,129.4, 147.5, 175.8; HMRS (ESI) calcd for [C₅₃H₈₇N₅O₁₅+H]⁺ 1034.6271.found 1034.6246.

Synthesis ofDescladinose-Clarithromycin-N-phenyltriazolylheptahydroxamic acid (26)

Reaction of descladinose-clarithromycin-N-phenylacetylene 21 (0.075 g,0.109 mmol) and 7-azidoheptahydroxamic acid 15b (0.040 g, 0.233 mmol)within 4 h, followed by prep TLC (silica, 10:1:0.1 CH₂Cl₂/MeOH/conc.NH₄OH) gave 80 mg (84%) of 26 as a brownish white solid. ¹H-NMR (CDCl₃,400 MHz) δ 0.78 (t, J=7.2 Hz), 1.06-1.31 (m), 1.40-1.53 (m), 1.71 (d,J=11.6 Hz), 1.80-1.91 (m), 2.01-2.20 (m), 2.50-2.65 (m), 2.91-2.97 (m),3.16 (t, J=6.4 Hz), 3.26-3.35 (m), 3.42-3.54 (m), 3.64-3.71 (m), 3.80(br s), 3.90 (br s), 4.30-4.34 (m), 5.12 (dd, J=11.6, 2.4 Hz), 7.27 (d,J=8.0 Hz), 7.72 (d, J=7.2 Hz), 7.80 (s); ¹³C NMR (CDCl₃, 100 MHz) δ 8.3,10.3, 12.5, 15.2, 16.1, 17.6, 18.6, 21.1, 21.3, 24.8, 25.1, 25.5, 26.2,27.8, 28.5, 29.1, 29.6, 29.7, 32.3, 35.8, 36.3, 37.4, 38.6, 44.3, 45.4,49.5, 50.0, 51.2, 57.8, 64.2, 69.7, 69.9, 70.6, 74.1, 77.9, 78.7, 88.0,106.3, 119.9, 125.7, 129.3, 129.5, 138.3, 147.3, 175.1; HMRS (ESI) calcdfor [C₄₅H₇₃N₅O₁₂+H]⁺ 876.5329. found 876.5301.

Synthesis of Clarithromycin-N-Phenyltriazolyloctahydroxamic acid (27)

Reaction of clarithromycin-N-phenylacetylene 20 (0.101 g, 0.120 mmol)and 8-azidooctahydroxamic acid 15e (0.047 g, 0.24 mmol) within 2.5 h,followed by prep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) gave 92mg (74%) of 27 as a brownish white solid. ¹H NMR (CDCl₃, 400 MHz) δ 0.81(t, J=7.2 Hz), 1.04-2.05 (m), 2.22 (s), 2.19-2.82 (m), 3.00 (s), 3.08(s), 2.91-3.80 (m), 3.95 (m), 4.38 (m), 4.88 (d, J=4.4 Hz), 5.04 (dd,J=10.8, 2.0 Hz), 7.33 (d, J=7.6 Hz), 7.71 (s), 7.77 (d, J=8.0 Hz); ¹³CNMR (CDCl₃, 400 MHz) δ 8.8, 9.4, 10.8, 12.5, 16.2, 18.2, 18.8, 20.0,21.2, 21.5, 21.6, 25.1, 26.0, 26.7, 28.2, 28.6, 28.9, 29.9, 30.1, 35.0,36.9, 37.4, 39.2, 39.4, 45.2, 45.4, 46.1, 49.6, 50.4, 50.8, 51.6, 58.1,63.6, 65.9, 68.4, 69.3, 70.9, 72.8, 74.4, 78.0, 78.5, 81.2, 96.1, 120.1,102.6, 126.1, 130.2, 147.4, 176.0; HRMS (ESI) calc for [C₅₄H₉₀N₅O₁₅+H]⁺1048.6427. found 1048.6486.

Synthesis of Descladinose-Clarithromycin-N-Phenyltriazolyloctahydroxamicacid (28)

Reaction of clarithromycin-N-phenylacetylene 21 (0.10 g, 0.144 mmol) and8-azidooctahydroxamic acid 15c (0.049 g, 0.246 mmol) within 2.5 h,followed by prep TLC (silica, 10:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) gave 117mg (90%) of 28 as a brownish white solid. ¹H NMR (CDCl₃, 400 MHz) δ 0.81(t, J=7.2 Hz), 1.10-2.09 (m), 2.18 (s), 2.19-2.68 (m), 2.98-3.73 (m),3.83 (s), 3.93 (m), 4.36 (m), 5.16 (d, J=8.0 Hz), 7.31 (d, J=8.0 Hz),7.77 (2H, d, J=8.0 Hz), 7.79 (1H, s); ¹³C NMR (CDCl₃, 400 MHz) δ 8.5,10.6, 12.8, 15.4, 16.4, 17.9, 18.9, 21.4, 21.6, 29.4, 36.1, 36.7, 37.7,38.9, 44.7, 45.7, 49.8, 58.0, 65.2, 70.0, 70.4, 70.8, 74.4, 76.8, 78.2,79.2, 83.6, 88.8, 106.9, 121.3, 128.7, 132.5, 139.6, 175.2; HRMS (FAB,thioglycerol) calc for [C₄₆H₇₆N₅O₁₂+H]⁺ 890.5490. found 890.5562.

Synthesis of Clarithromycin-N-Phenyltriazolylnonahydroxamic acid (29)

Reaction of clarithromycin-N-phenylacetylene 20 (0.100 g, 0.120 mmol)and 9-azidononahydroxamic acid 15d (0.043 g, 0.20 mmol) within 2.5 h,followed by prep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) gave 54mg (42%) of 29 as a brownish white solid. ¹H NMR (CDCl₃, 400 MHz) δ 0.81(t, J=7.2 Hz), 1.04-2.02 (m), 2.24 (s), 2.10-2.97 (m), 3.00 (s), 3.09(s), 3.20-3.82 (m), 3.88 (m), 4.39 (m), 4.88 (d, J=4.0 Hz), 5.05 (d,J=10.0 Hz), 7.35 (d, J=8.0 Hz), 7.77 (d, J=4.0 Hz); ¹³C NMR (CDCl₃, 400MHz) δ 9.0, 9.3, 10.8, 12.5, 16.1, 18.2, 18.8, 21.2, 21.5, 21.6, 21.7,21.8, 26.3, 26.8, 28.7, 28.9, 29.1, 29.9, 30.3, 35.0, 37.0, 37.4, 39.3,39.4, 45.2, 45.4, 49.6, 50.5, 50.8, 51.6, 57.8, 63.8, 65.8, 68.8, 69.2,70.9, 72.7, 74.5, 76.8, 78.1, 78.4, 78.5, 81.1, 96.1, 102.9, 119.7,125.9, 129.6, 129.9, 138.7, 147.6, 176.1; HRMS (ESI) calc for[C₅₅H₉₁N₅O₁₅+H]⁺ 1062.6584. found 1062.6586.

Synthesis of Clarithromycin-N-Phenyltriazolyldecahydroxamic acid (30)

Reaction of clarithromycin-N-phenylacetylene 20 (0.10 g, 0.120 mmol) and10-azidodecahydroxamic acid 15e (0.045 g, 0.197 mmol) within 2.5 h,followed by prep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) gave 68mg (53%) of 30 as a brownish white solid. ¹H NMR (CDCl₃, 400 MHz) δ 0.82(t, J=7.2 Hz), 1.05-2.12 (m), 2.24 (s), 2.26-2.97 (m), 3.01, 3.10 (s),3.19-3.80 (m), 3.95 (m), 4.39 (m), 4.89 (d, J=4.0 Hz), 5.04 (d, J=8.0Hz), 7.35 (d, J=8.0 Hz), 7.76 (s), 7.79 (d, J=8.0 Hz); ¹³C NMR (CDCl₃,400 MHz) δ 9.3, 10.8, 12.5, 16.1, 18.2, 18.8, 20.0, 21.2, 21.5, 21.7,25.4, 26.2, 28.9, 29.0, 29.3, 29.6, 29.9, 30.2, 35.0, 37.0, 37.4, 39.3,39.4, 45.2, 45.4, 49.6, 50.5, 50.8, 51.6, 57.8, 63.8, 65.8, 68.8, 69.2,70.9, 72.7, 74.5, 76.8, 78.1, 78.5, 81.1, 96.1, 102.9, 119.7, 125.9,129.6, 129.8, 138.9, 147.6, 176.1; HRMS (ESI) calc for [C₅₆H₉₃N₅O₁₅+H]⁺1076.6740. found 1076. 6667.

Example 2 Synthesis of Compounds 36 and 38 in Table 3 Synthesis ofMethyl 8-(4-(hydroxymethyl)phenylamino)-8-oxooctanoate (33)

To a mixture of 8-methoxy-8-oxooctanoic acid (0.40 g, 2.10 mmol),benzotriazole (0.28 g, 2.23 mmol) in anhydrous CH₂Cl₂ (15 mL) was addedSOCl₂ (0.17 mL, 2.23 mmol) at 0° C., the mixture was kept stirring at 0°C. for 2.5 h and then filtered. The solvent was evaporated off to givecrude acid chloride 31 which was used without further purification.

To a solution of (4-aminophenyl)methanol 32 (0.31 g, 2.50 mmol) inanhydrous pyridine (8 mL) was added chlorotrimethylsilane (0.32 mL, 2.50mmol) at room temperature and stirring continued for 2 h. The mixture,together with a catalytic amount of DMAP, was added to a mixture ofcrude chloride 31 (obtained as described above) in pyridine at 0° C. Thereaction was allowed to warm to room temperature and stirring continuedovernight. Water (5 mL) and 1 M TBAF in tetrahydrofuran (THF) (0.25 mL,0.25 mmol) were added and stirring continued for additional 30 min.EtOAc (50 mL) and 1N HCl (30 mL) were added, the two layers wereseparated, the organic layer was washed with 1N HCl (30 mL) andsaturated brine (30 mL) and dried over Na₂SO₄. Solvent was evaporatedoff and the crude was purified by preparative TLC, eluting withacetone/hexanes 1:1 to give compound 33 (195 mg, 30%) as yellow-whitesolid. ¹H-NMR (CDCl₃, 400 MHz) δ 1.24 (4H, m), 1.49-1.59 (4H, m),2.17-2.25 (4H, m), 3.58 (3H, s), 4.50 (2H, s), 7.13 (2H, d, J=8.4 Hz),7.37 (2H, d, J=8.4 Hz).

Synthesis of Azithromycin-arylalkyl methyl ester (35)

To a solution of crude preparation of compound 33 (0.64 g, 2.20 mmol) inCH₂Cl₂ (15 mL) and triethylamine (Et₃N) (0.90 mL, 6.60 mmol) was addedmesyl chloride (0.70 mL, 8.85 mmol) at 0° C. and the reaction wasallowed to warm to room temperature. Stirring continued for 2 h, CH₂Cl₂(40 mL) and saturated sodium bicarbonate (30 mL) were added. The twolayers were separated; the organic layer was washed with sodiumbicarbonate (1×30 mL), saturated brine (30 mL) and dried over Na₂SO₄.Solvent was evaporated off and the crude was purified by flashchromatography (silica gel, eluting with Hexane/EtOAc, gradient 3:1,2:1, 1:1) to give compound 34 (320 mg, 40%) as white solid.

A mixture of 4′-Desmethylazithromycin 1 (0.45 g, 0.62 mmol), compound 34(0.32 g, 0.86 mmol), catalytic amount of potassium iodide in THF (15 mL)and Hunig's base (3 mL) was heated under refluxing condition for 48 h.CH₂Cl₂ (80 mL) and saturated sodium bicarbonate (40 mL) were added andthe two layers were separated. The organic layer was washed with sodiumbicarbonate (40 mL), saturated brine (40 mL) and dried over Na₂SO₄.Solvent was evaporated off and the crude was purified by preparativeTLC, eluting with EtOAc/hexanes/Et₃N 3:2:0.1 to give compound 35 (176mg, 28%) as brown-white solid. ¹H-NMR (CDCl₃, 400 MHz) δ 0.79-0.83 (m),0.94-1.01 (m), 1.09-1.20 (m), 1.22-1.32 (m), 1.37-1.59 (m), 1.62-1.73(m), 1.77-2.01 (m), 2.08-2.28 (m), 2.38-2.52 (m), 2.61-2.71 (m),2.91-3.01 (m), 3.11 (s), 3.25-3.33 (m), 3.42 (m), 3.54-3.65 (m), 3.97(m), 4.18 (m), 4.36 (d, J=6.8 Hz), 4.61 (m), 5.03 (d, J=4.4 Hz), 7.14(d, J=8.4 Hz), 7.42 (d, J=8.4 Hz), 7.73 (s), 8.97 (bs); ¹³C-NMR (CDCl₃,100 MHz) δ 7.5, 9.1, 11.3, 14.8, 16.2, 16.9, 18.2, 20.5, 21.2, 21.4,21.5, 21.9, 24.6, 25.3, 26.7, 27.5, 28.7, 29.6, 33.9, 34.7, 36.2, 36.7,37.3, 39.1, 41.9, 42.2, 45.1, 48.5, 49.3, 51.4, 57.3, 62.2, 64.3, 65.4,68.5, 69.9, 70.5, 72.7, 73.5, 73.8, 74.1, 77.7, 77.9, 83.4, 94.4, 102.6,119.5, 129.0, 134.2, 137.0, 171.0, 173.8, 178.4. MS (FAB, mnba) 1010.3(M+H)⁺.

Synthesis of Azithromycin-arylalkyl hydroxamic acid (36)

To a solution of compound 35 (0.09 g, 0.09 mmol) in 1:1 THF/MeOH (3 mL)was added hydroxylamine (50% in H₂O) (0.03 mL, 0.54 mmol) and acatalytic amount of KCN. The mixture was stirred at room temperature for24 h. The reaction was partitioned between 5% MeOH in CH₂Cl₂ (30 mL) andsaturated sodium bicarbonate (25 mL), the two layers were separated andthe aqueous layer was extracted with 5% MeOH in CH₂Cl₂ (2×20 mL). Thecombined organic layer was washed with saturated brine (40 mL) and driedover Na₂SO₄. Solvent was evaporated off and the crude was purified bypreparative TLC, eluting with CH₂Cl₂/MeOH/NH₄OH 10:1:0.1 to givecompound 36 (22 mg, 25%) as brown-white solid. ¹H-NMR (CD₃OD, 400 MHz) δ0.87-0.92 (m), 1.02-1.12 (m), 1.17-1.37 (m), 1.43-1.69 (m), 1.75-1.88(m), 1.99 (m). 2.08 (m), 2.13-2.19 (m), 2.24 (s), 2.30 (s), 2.33-2.41(m), 2.54 (d, J=11.2 Hz), 2.75-2.80 (m), 3.00 (d, J=9.6 Hz), 3.19 (bs),3.47-3.51 (m), 3.60 (bs), 3.63-3.78 (m), 4.14-4.22 (m), 4.50 (d, J=7.2Hz), 5.02 (d, J=4.8 Hz), 7.29 (d, J=8.0 Hz), 7.49 (d, J=8.4 Hz). MS(FAB, mnba) 1011.3 (M+H)⁺.

Synthesis of Desclasinose-azithromycin-arylalkyl hydroxamic acid (38)

A mixture of compound 35 (0.05 g, 0.05 mmol) in 0.25 N HCl (15 mL) wasstirred at room temperature for 20 h and poured into EtOAc (20 mL). Thetwo layers were separated and the aqueous layer was washed with EtOAc(2×20 mL), basified with concentrated NH₄OH and then extracted with 5%MeOH in CH₂Cl₂ (2×30 mL). The combined organic layer was washed withsaturated brine (30 mL) and dried over Na₂SO₄. Solvent was evaporatedoff to give compound 37 which was used for the next reaction withoutfurther purification.

To a solution of compound 37 (obtained as described above) in 1:1THF/MeOH (2 mL) was added hydroxylamine (50% in H₂O) (0.05 mL, 0.79mmol) and a catalytic amount of KCN. The mixture was stirred at roomtemperature for 24 h. The reaction was partitioned between 5% MeOH inCH₂Cl₂ (30 mL) and saturated brine (20 mL), the two layers wereseparated and the organic layer was dried over Na₂SO₄. Solvent wasevaporated off and the crude was purified by preparative TLC, elutingwith CH₂Cl₂/MeOH/NH₄OH 9:1:0.1 to give compound 38 (7 mg, 16%) asbrown-white solid. ¹H-NMR (CD₃OD, 400 MHz) δ 0.78 (m), 0.85 (d, J=7.2Hz), 0.91 (d, J=8.0 Hz), 0.99 (s), 1.11 (m), 1.26-1.76 (m), 1.98 (t,J=7.4 Hz), 2.08 (m), 2.17 (s), 2.25 (t, J=7.4 Hz), 2.40 (bs), 2.58 (m),2.95 (bs), 3.24 (m), 3.37-3.56 (m), 3.67 (d, J=13.2 Hz), 4.53 (d, J=7.6Hz), 7.19 (d, J=8.4 Hz), 7.39 (d, J=8.4 Hz). MS (FAB, mnba) 853.3(M+H)⁺.

Example 3 Synthesis of Compounds 40, 44 and 47 in Table 3 Synthesis ofAzithromycin-N-phenyltriazolylhepta-2-methyl ketone (40)

Compound 3 (0.040 g, 0.047 mmol) and azido-2-methyl ketone 39 (0.011 g,0.071 mmol) were dissolved in anhydrous THF (7 mL) and stirred underargon at room temperature. Copper (I) iodide (0.010 g, 0.053 mmol) andHunigs' base (0.1 mL) were then added to reaction mixture and stirringcontinued for 2 h. The reaction mixture was diluted with CH₂Cl₂ (40 mL)and washed with 1:4 NH₄OH/saturated NH₄Cl (3×30 mL) and saturated NH₄Cl(30 mL). The organic layer was dried over Na₂SO₄ and concentrated invacua. The crude product was purified by preparative TLC (12:1CH₂Cl₂:MeOH) to give 38 mg (81%) of 40 as a white solid. ¹H NMR (CDCl₃,400 MHz) δ 0.86-0.89 (t, J=7.2 Hz), 0.92-0.98 (m), 1.01-1.02 (d, J=7.2Hz), 1.086 (s), 1.12-1.24 (m), 1.29-1.34 (m), 1.42-1.66 (m), 1.74-1.78(d, J=16 Hz), 1.84-2.08 (m), 2.11 (s), 2.15 (m), 2.26-2.48 (m),2.56-2.74 (m), 2.84 (s), 2.74-3.08 (m), 3.11 (s), 3.32-3.40 (m),3.46-3.84 (m), 3.98-4.46 (m), 4.23 (broad singlet), 4.38-4.51 (m), 4.72(broad singlet), 5.06-5.12 (m), 7.31-7.38 (m), 7.75 (s), 7.77-7.79 (d,J=8 Hz); HRMS (ESI) calc for [C₅₃H₈₉N₅O₁₃+H]⁺ 1004.6529. found1004.6482.

Synthesis of Azidobenzamide (43)

A solution of azido acid 41 (0.150 g, 0.877 mmol) in dry THF (10 mL) wastreated with 1,2-diaminobenzene 42 (0.568 g, 5.26 mmol) and EDC (0.219g, 1.14 mmol). The resulting mixture was stirred at room temperature forabout 24 h and then concentrated in vacua. The crude was diluted withEtOAc (40 mL), washed in succession with water (30 mL) and brine (30 mL)and the organic layer was dried over Na₂SO₄. Solvent was evaporated offand the crude was purified by flash chromatography (silica,Hexanes/EtOAc 1:2) to give 79 mg (35%) of 43 as a yellow solid. ¹H NMR(CDCl₃, 400 MHz) δ 1.34-1.68 (m), 2.27-2.31 (m), 3.84 (s), 6.72-6.75(m), 7.00-7.05 (m), 7.09-7.11 (d, J=8 Hz); HRMS (ESI) calcd for[C₁₃H₁₉N₅O+H]⁺ 262.1662. found 262.1635.

Synthesis of Azithromycin-N-phenyltriazolylheptabenzamide (44)

Compound 3 (0.050 g, 0.059 mmol) and azido benzamide 43 (0.023 g, 0.088mmol) were dissolved in anhydrous THF (10 mL) and stirred under argon atroom temperature. Copper (I) iodide (0.010 g, 0.0526 mmol) and Hunigs'base (0.1 mL) were then added to reaction mixture and stirring continuedfor 2 h. The reaction mixture was diluted with CH₂Cl₂ (40 mL), washedwith 1:4 NH₄OH/saturated NH₄Cl (3×30 mL) and saturated NH₄Cl (30 mL).The organic layer was dried over Na₂SO₄ and concentrated in vacuo. Thecrude product is purified by prep TLC (12:1 CH₂Cl₂/MeOH) to give 38 mg(59%) of 44 as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 0.80-1.57 (m),1.70-1.75 (m), 1.85-1.91 (m), 2.01-2.07 (m), 2.13 (s), 2.22-2.26 (m),2.36-2.41 (m), 2.58 (br s), 2.68 (br s), 2.75-3.07 (m), 3.25-3.62 (m),3.69 (s), 3.81 (br s), 3.98 (br s), 4.17 (s), 4.32-4.48 (m), 4.72 (br s)5.05 (s), 6.70-6.76 (m), 6.92-7.02 (m), 7.18-7.21 (d, J=12 Hz), 7.32 (brs), 7.71-7.73 (d, J=8 Hz), 7.78 (s). 7.87 (br s); HRMS (ESI) calcd for[C₅₉H₉₅N₇O₁₃+H]⁺ 1110.7060. found 1110.7012.

Synthesis of Descladinose-clarithromycin-N-phenylacetylene-O-Acetate(45)

Descladinose-clarithromycin-N-phenylacetylene 21 (3.80 g, 5.5 mmol) wasdissolved in acetone (20 ml) followed by addition of acetic anhydride(0.62 g, 6.0 mmol) and stirred at 40° C. for 36 h. The reaction mixturewas diluted with EtOAc (100 mL), washed with aqueous NaHCO₃ and brine,and then purified on silica column eluting with 6:1 CH₂Cl₂/Acetone toobtain 2.8 g (70%) of 45 as a brownish white solid. ¹H NMR (CDCl₃, 400MHz) δ 0.80 (t, J=7.2 Hz), 0.90 (d, J=7.2 Hz), 1.08-1.47 (m), 1.58 (s),1.63-2.05 (m), 2.08 (s), 2.16 (s), 2.42-2.80 (m), 2.92 (s), 2.94-3.00(m), 3.03-3.68 (m), 3.79 (s), 3.94 (s), 4.08 (m), 4.54 (d, J=8.0 Hz),4.80 (m), 5.15 (dd, J=11.6, 2.4 Hz), 7.17 (d, J=8.4 Hz), 7.38 (d, J=8.0Hz).

Synthesis of clarithromycin-N-phenylacetylene ketolide (46)

Methyl sulfide (0.35 g, 5.7 mmol), was added to a mixture ofN-chlorosuccinimide (0.65 g, 4.8 mmol) and CH₂Cl₂ (3 mL) whilemaintaining the temperature at −15° C. Compound 45 (2.5 g, 3.4 mmol)dissolved in CH₂Cl₂ (20 mL) was added to the reaction mixture, followedby triethylamine (0.39 g, 3.8 mmol). The mixture was stirred at −15° C.for 3.5 h and partitioned between EtOAc (100 mL) and 0.5 N aqueous NaOH(150 mL). The organic layer was separated, washed with brine (70 mL),and dried over Na₂SO₄. Solvent was evaporated off and the crude waspurified on silica column eluting with 1:4:0.1 EtOAc/Hexane/Et₃N,increasing solvent polarity to 2:3:0.1, to afford 2.0 g (80%) of 46 asoff-white solid. ¹H NMR (CDCl₃, 400 MHz) δ 0.80-0.86 (m), 1.09-1.57 (m),1.58 (s), 1.62-2.02 (m), 2.05 (s), 2.15 (s), 2.44-2.80 (m), 2.92 (s),2.95-3.00 (m), 3.05-3.82 (m), 4.12 (m), 4.38 (d, J=8.0 Hz), 4.79-4.83(m), 5.14 (dd, J=11.2, 2.0 Hz), 7.16 (d, J=7.6 Hz), 7.38 (d, J=7.6 Hz).

Synthesis of Ketolide-N-phenyltriazolylheptabenzamide (47)

Ketolide 46 (0.050 g, 0.069 mmol) and azido benzamide 43 (0.027 g, 0.103mmol) were dissolved in anhydrous THF (10 mL) and stirred under argon atroom temperature. Copper (I) iodide (0.010 g, 0.0526 mmol) and Hunigs'base (0.1 mL) were then added to reaction mixture and stirring continuedfor 2 h. The reaction mixture was diluted with CH₂Cl₂ (40 mL) and washedwith 1:4 NH₄OH/saturated NH₄Cl (3×30 mL) and saturated NH₄Cl (30 mL).The organic layer was dried over Na₂SO₄ and concentrated in vacuo. Thecrude was purified by prep TLC (3:2 CH₂Cl₂/Acetone) to give 51 mg (75%)of 47 as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 0.78-0.88 (m),1.18-1.25 (m), 1.31-1.70 (m), 1.87-2.00 (m), 2.14 (s), 2.28-2.35 (m),2.42-2.51 (m), 2.62-2.75 (m), 3.12 (s), 3.31-3.35 (t, J=8 Hz), 3.42 (s),3.47 (br s), 3.62-3.82 (m), 4.32-4.40 (m), 5.05-5.09 (d, J=16 Hz), 5.44(s), 5.77 (s), 6.64-6.74 (m), 6.95-7.00 (m), 7.10-7.15 (m), 7.18-7.21(m), 7.27-7.31 (m), 7.60 (s) 7.72-7.74 (d, J=8 Hz), 7.77 (s).

Example 4 Synthesis of Compounds 56a-e

The procedure for the synthesis of compounds 56a-e as shown in Scheme 16is described below.

4′-Desmethylclarithromycin (2)

To a solution of clarithromycin 1 (50.0 g, 68.1 mmol) and sodium acetate(50.3 g, 61.3 mmol) in 80% aqueous methanol (350 mL) at 75-80° C. wasadded iodine (19.0 g, 74.8 mmol) in three batches within 5 min. Thereaction was maintained at pH 8-9 by additions of 1M NaOH (2×50 ml, onceat 10 min and 45 min of reaction time). Stirring was continued at 80° C.for 4.5 h. A solution of 5% sodium thiosulfate (300 mL) anddichloromethane (250 mL) were added and the two layers were separated.The aqueous layer was extracted with CH₂Cl₂ (150 mL), the combinedorganic layers were washed with saturated brine, dried over Na₂SO₄, andconcentrated in vacuo to give 44.0 g of 2, which was used withoutfurther purification.

4-Ethynylbenzyl methanesulfonate (3)

To a solution of 4-ethynylbenzyl alcohol (5.0 g, 37.8 mmol) in CH₂Cl₂(50 mL) and triethylamine (Et₃N) (14.4 mL, 104.0 mmol) was added mesylchloride (8.04 mL, 104.0 mmol) at 0° C. and the reaction was allowed towarm to room temperature. Stirring continued for 2 h under argon duringwhich TLC revealed a quantitative conversion into a higher Rf product.Ice cold water was added into the reaction mixture and extracted withEt₂O (200 ml). The organic layer was washed with 1N HCL (70 ml), H₂O(100 ml), aqueous NaHCO₃ (70 ml), and finally H₂O (100 ml); and thendried over Na₂SO₄. Solvent was evaporated off and dried in vacuo to give7.0 g of compound 3 as brownish oil which was used without furtherpurification.

4′-Ethynylbenzylclarithromycin (4)

To a solution of 4′-desmethylclarithromycin 2 (2.4 g, 3.34 mmol) inanhydrous DMSO (4 ml), was added Hunig's base (3 ml), and4-ethynylbenzyl methanesulfonate 2 (0.92 g, 4.34 mmol). The reactionmixture was heated at 85° C. for 2.5 h. The reaction was cooled anddiluted with EtOAc (30 ml), then washed with saturated NaHCO₃ (3×10 ml)and saturated brine (10 ml). The organic layer was dried over Na₂SO₄ andthe solvent evaporated. The crude product was purified on silica columnusing 12:1 CH₂Cl₂/C₂H₆CO gradually increasing the solvent polarity to10:1 and 8:1 to afford 1.8 g (63%) of the title compound.

¹H-NMR (CDCl₃, 400 MHz) δ 0.82 (3H, t, J=7.2 Hz), 1.03-1.28 (18H, m),1.37 (3H, s), 1.40-1.55 (3H, m), 1.65-1.90 (6H, m), 2.03 (1H, d, J=10.0Hz), 2.22 (3H, s), 2.30 (1H, d, J=15.2 Hz), 2.40-2.60 (2H, m), 2.80-2.90(2H, m), 294-3.00 (6H, m), 3.04 (1H, s), 3.09 (3H, s), 3.16 (1H, s),3.24-3.29 (1H, m), 3.38-3.46 (3H, m), 3.59 (1H, d, J=6.8 Hz), 3.70-3.75(3H, m), 3.88-3.95 (1H, m), 4.37 (1H, d, J=7.2 Hz), 4.88 (1H, d, J=4.4Hz), 5.02 (1H, dd, J=11.6, 2.4 Hz), 7.23 (2H, d, J=12.0 Hz), 7.42 (2H,d, J=8.0 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 9.2, 10.7, 12.4, 16.1, 18.1,18.7, 19.9, 21.1, 21.5, 29.3, 32.4, 34.8, 36.9, 37.2, 39.2, 45.0, 45.2,49.3, 50.6, 53.4, 57.6, 63.3, 65.6, 68.5, 69.0, 70.6, 72.5, 74.2, 76.5,77.8, 78.1, 78.2, 80.8, 95.8, 102.5, 120.9, 128.6, 132.0, 133.5, 139.4,175.4; HRMS (ESI) talc for [C46H73NO13+H]+ 848.5155. found 848.5181

Descladinose-4′-Ethynylbenzylclarithromycin (5)

A solution of 4′-Ethynylbenzylclarithromycin 4 (27.3 g, 31.2 mmol) inEtOH (200 ml) and 1N HCL (200 ml) was stirred at room temperature for 17h after which TLC analysis indicated the absence of starting material.The reaction mixture was basified with NH4OH to about pH 9, added H₂O(250 ml) and EtOAc (350 ml) and separated the two layers. The aqueouslayer was washed with EtOAc (250 ml) and the two organic layers combinedand washed with saturated brine, then dried over Na₂SO₄. The crudeproduct was purified on silica column using gradient elution (12:1, 10:1and 8:1 CH2Cl/2/C₂H₆CO) to isolate 77% (17.1 g) of 5 as off-white foam.

¹H-NMR (CDCl₃, 400 MHz) δ 0.82 (3H, t, J=7.6 Hz), 1.09-1.28 (12H, m),1.34 (3H, s), 1.40-1.55 (3H, m), 1.70-1.74 (2H, m), 1.87-1.94 (3H, m),2.08-2.15 (6H, m), 2.54-2.66 (2H, m), 2.94-2.98 (3H, m), 3.05 (1H, s),3.25 (1H, s), 3.31-3.42 (2H, m), 3.48-3.56 (2H, m), 3.66 (2H, d, J=10.0Hz), 3.82 (1H, s), 3.90 (1H, s), 4.35 (1H, d, J=7.6 Hz), 5.14 (1H, dd,J=10.8, 2.0 Hz), 7.18 (2H, d, J=8.0 Hz), 7.42 (2H, d, J=8.4 Hz); ¹³C NMR(CDCl₃, 100 MHz) δ 8.4, 10.5, 12.7, 15.3, 16.3, 17.8, 18.8, 21.4, 29.2,35.9, 36.6, 37.5, 38.7, 44.5, 45.5, 49.6, 57.8, 65.0, 69.7, 70.1, 70.6,74.1, 77.9, 78.9, 83.3, 88.5, 106.5, 121.0, 128.4, 132.1, 139.1, 174.7;HRMS (ESI) calc for [C38H59NO10+H]+ 690.4212. found 690.4259

Synthesis of compound (6) (Acetylation)

To a solution of descladinose-4′-ethynylbenzylclarithromycin 5 (3.8 g,5.5 mmol) in acetone (20 ml), Ac₂O (0.62 g, 6.0 mmol) was added andstirred at 40° C. for 36 h. The reaction mixture was diluted with EtOAc(100 ml) and washed with aqueous NaHCO₃ (70 ml) and saturated brine (70ml). Purification on silica column (6:1 CH₂Cl₂/acetone) afforded 2.8 g(70%) of the title compound as a yellowish solid.

¹H NMR (CDCl₃, 400 MHz) δ 0.80 (3H, t, J=7.2 Hz), 0.90 (3H, d, J=7.2Hz), 1.08-1.47 (12H, m), 1.58 (6H, s), 1.63-2.05 (7H, m), 2.08 (3H, s),2.16 (3H, s), 2.42-2.80 (3H, m), 2.92 (3H, s), 2.94-3.00 (1H, m),3.03-3.68 (3H, m), 3.79 (2H, s), 3.94 (1H, s), 4.08 (1H, m), 4.54 (1H,d, J=8.0 Hz), 4.80 (1H, m), 5.15 (1H, dd, J=11.6, 2.4 Hz), 7.17 (2H, d,J=8.4 Hz), 7.38 (2H, d, J=8.0 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 8.0, 10.7,12.8, 15.5, 16.4, 18.1, 19.5, 21.3, 21.5, 21.6, 31.1, 31.9, 36.0, 37.0,37.5, 38.7, 44.3, 45.7, 50.0, 58.4, 62.5, 68.9, 69.8, 71.6, 74.4, 76.9,77.0, 77.8, 78.1, 81.3, 83.9, 99.9, 120.7, 128.4, 132.2, 141.1, 170.1,174.9, 221.2; HRMS (FAB, mnba) calc for [C40H61NO11+H]+ 732.43229. found732.43105

Synthesis of Compound (7) (Oxidation)

Methyl sulfide (1.42 g, 22.8 mmol), was added to a mixture ofN-chlorosuccinimide (2.61 g, 19.5 mmol) and CH₂Cl₂ (10 ml) whilemaintaining the temperature at −15° C. Compound 6 (10.0 g, 13.7 mmol)dissolved in CH₂Cl₂ (50 ml) was added to the reaction flask, followed byEt₃N (1.56 g, 15.4 mmol) and stirred at −15° C. for 4.5 h. The reactionmixture was poured into EtOAc (350 ml) and 0.5 N aqueous NaOH (250 ml).The organic layer was separated and washed with saturated brine (250ml), dried over Na₂SO₄, evaporated solvent and purified on silica column(2:3:0.1 EtOAc/Hexane/Et₃N) to afford 9.54 g (96%) of the title compoundas off-white solid.

¹H NMR (CDCl₃, 400 MHz) δ 0.80-0.86 (6H, m), 1.09-1.57 (12H, m), 1.58(6H, s), 1.62-2.02 (7H, m), 2.05 (3H, s), 2.15 (3H, s), 2.44-2.80 (2H,m), 2.92 (3H, s), 2.95-3.00 (1H, m), 3.05-3.66 (4H, m), 3.80 (1H, q,J=14.0, 6.8 Hz), 3.89 (1H, s), 4.12 (1H, m), 4.38 (1H, d, J=8.0 Hz),4.79-4.83 (1H, m), 5.14 (1H, dd, J=11.2, 2.0 Hz), 7.16 (2H, d, J=7.6Hz), 7.38 (2H, d, J=7.6 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 10.8, 12.4,14.4, 14.5, 16.5, 17.9, 19.6, 21.3, 21.5, 31.3, 36.9, 37.6, 39.2, 45.1,46.3, 49.7, 51.1, 58.5, 62.5, 69.2, 69.6, 71.5, 74.1, 77.0, 77.1, 77.2,78.1, 83.9, 101.1, 120.7, 128.5, 132.2, 140.9, 169.6, 170.0, 205.7,221.1; HRMS (FAB, mnba) talc for [C40H59NO11+H]+ 730.41664. found730.41321

Carbamate Ketolide (8)

To a suspension of 7 (1.5 g, 2.06 mmol) in a mixture of anhydrous THF(20 ml) and anhydrous DMF (7 ml), was added CDI (1.3 g, 8.20 mmol)followed by a solution of NaN(TMS)₂ 1.0 M in THF (2.6 ml, 2.6 mmol) over75 min and stirred at RT for 24 h. The reaction mixture was diluted withEtOAc (50 ml), washed with aqueous NaHCO₃ (20 ml) and saturated brine(20 ml) and dried over Na₂SO₄. The solvent was evaporated and purifiedon silica column (3:2:0.1 EtOAc/Hexane/Et3N) to yield 42% (0.7 g) of thetitle compound. NMR (CDCl₃, 400 MHz) δ 0.91 (3H, t, 7.6 Hz), 1.09 (3H,d, J=7.2 Hz), 1.14-1.50 (9H, m), 1.80 (3H, s), 1.83 (3H, s), 1.56-1.94(7H, m), 2.00 (3H, s), 2.05 (3H, s), 2.12 (3H, s), 2.64-2.80 (1H, m),3.02 (3H, s), 2.95-3.70 (4H, m), 3.73 (1H, q, J=14.0, 6.8 Hz), 4.10-4.06(2H, m), 4.30 (1H, d, J=7.6 Hz), 4.77-4.80 (1H, m), 5.68 (1H, dd,J=11.2, 2.0 Hz), 6.77 (1H, s), 7.03 (1H, s), 7.16 (2H, d, J=7.6 Hz),7.36 (3H, m), 8.06 (1H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 10.7, 13.5, 14.4,15.2, 19.1, 20.4, 21.1, 21.2, 21.5, 22.8, 31.2, 36.8, 47.6, 50.5, 51.2,58.5, 60.6, 62.8, 69.3, 71.6, 78.7, 83.9, 84.7, 117.3, 120.7, 128.4,131.1, 132.2, 137.3, 138.8, 140.9, 146.2, 169.1, 169.8; HRMS (FAB, mnba)calc for [C44H59N3O11+H]+ 806.42279. found 806.42103

Tricyclic Ketolide (9)

To a solution of 8 (0.6 g, 0.74 mmol) in CH₃CN (10 ml) and H₂O (1 ml),was added ethylenediamine (0.44 g, 7.40 mmol) and heated in a sealedtube at 50-55° C. for 24 h. The solvent was evaporated off and thereaction mixture partitioned between H₂O (15 ml) and CH₂Cl₂ (20 ml) andseparated. The organic layer was washed with saturated brine (20 ml),dried over Na₂SO₄, and the solvent evaporated. The crude product wasdissolved in anhydrous MeOH (15 ml) and heated at 90° C. in a sealedtube for 24 h. MeOH was evaporated and the product purified on silicacolumn (EtOAc/Et₃N 12:0.1). EtOH (6 ml) followed by AcOH (0.043 g, 0.71mmol) were added to the pure product (0.27 g, 0.34 mmol) and heated at95° C. in a sealed tube for 20 h. The reaction mixture was suspended indilute NH₄OH (10 ml) and CH₂Cl₂ (15 ml) and separated the organic layer,washed with saturated brine (10 ml), and dried over Na₂SO₄. Purificationon silica column (EtOAc/MeOH/Et₃N 10:0.5:0.05) afforded 0.24 g (57%) ofcompound 9.

¹H NMR (CDCl₃, 400 MHz) δ 0.80 (3H, t, J=7.2 Hz), 1.00 (3H, d, J=6.8Hz), 1.14-1.55 (12H, m), 1.30 (3H, s), 1.43 (3H, s), 1.60-1.96 (6H, m),2.12 (3H, s), 2.45-2.95 (6H, m), 3.03 (3H, s) 3.23-3.43 (4H, m),3.64-3.78 (4H, m), 3.94 (1H, m), 4.16 (1H, d, J=8.4 Hz), 4.25 (1H, d,J=7.6 Hz), 4.90 (1H, d, J=10.0 Hz), 7.18 (2H, d, J=8.0 Hz), 7.39 (2H, d,J=7.2 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 10.6, 11.1, 13.0, 14.6, 16.6,19.3, 19.8, 21.4, 22.3, 29.7, 36.5, 37.1, 38.7, 42.5, 43.0, 48.3, 49.3,49.7, 51.4, 57.6, 60.1, 65.6, 69.7, 70.5, 76.7, 78.7, 79.4, 81.7, 83.6,104.0, 121.0, 128.8, 132.4, 140.0, 156.3, 169.8, 181.5, 204.4; HRMS(FAB, mnba) talc for [C41H59N3O9+H]+ 738.43296. found 738.43318

Azidoalkyl-O-trityl Hydroxamate Derivatives (10a-e)

Method A

Tricyclic Ketolide-N-benzyltriazolylhexa-N-Otrityl (11a)

General Procedure: 4′-Ethynylbenzyl tricyclic ketolide 9 (0.105 g, 0.142mmol) and 6-azidohexa-o-trityl hydroxamic acid 10a (0.058 g, 0.142 mmol)were dissolved in anhydrous THF (7 mL) and stirred under argon at roomtemperature. Copper (I) iodide (0.012 g, 0.063 mmol) and Hunig's base(0.6 mL) were added to the reaction mixture, and stirring continued for24 h. The reaction mixture was diluted with 1:4 NH₄OH/saturated NH₄Cl(50 mL) and extracted with 10% MeOH/CH₂Cl₂ (3×30 mL). The organic layerswere combined, dried over Na₂SO₄ and concentrated in vacuo. The crudeproduct was purified by prep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/conc.NH₄OH) to give 127 mg (78%) of 11a as a yellowish solid.

¹H NMR (CDCl₃, 400 MHz) δ 0.85 (3H, t, J=7.6 Hz), 1.1 (3H, d, J=6.8 Hz),1.20-1.39 (17H, m), 1.46-2.00 (15H, m), 2.19 (3H, s), 2.58-2.80 (6H, m),2.84-3.00 (1H, m), 3.02-3.17 (1H, m), 3.31-3.60 (3H, m), 3.64-3.87 (5H,m), 3.96-4.0 (1H, m), 4.22 (1H, d, J=8.4 Hz), 4.29-4.32 (3H, m), 4.93(1H, dd, J=10.0, 1.6 Hz), 7.23-7.45 (17H, m), 7.71 (1H, s), 7.79 (2H, d,J=8.0 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 10.7, 11.1, 13.1, 14.6, 16.7,19.3, 19.9, 21.4, 22.3, 22.8, 26.0, 29.6, 30.2, 31.0, 36.5, 37.0, 38.8,42.5, 43.0, 48.3, 49.3, 49.7, 50.2, 51.4, 53.7, 57.7, 60.1, 65.6, 69.8,70.5, 76.7, 78.7, 79.4, 81.8, 93.1, 104.1, 119.7, 126.0, 128.2, 128.4,129.2, 129.4, 130.0, 138.9, 141.2, 147.6, 156.3, 169.8, 177.0, 181.5,204.5; HRMS (ESI) talc for [C66H85N7O11+H]+ 1152.6379. found 1152.6367

Tricyclic Ketolide-N-benzyltriazolylhepta-N-Otrityl (11b)

Reaction of 4′-ethynylbenzyl tricyclic ketolide 9 (0.102 g, 0.138 mmol)and 6-azidohepta-o-trityl hydroxamic acid 10b (0.059 g, 0.138 mmol)within 24 h as described for the synthesis and purification of 11a gave133 mg (83%) of 11b as a yellowish solid.

¹H NMR (CDCl₃, 400 MHz) δ 0.85 (3H, t, J=7.6 Hz), 1.06 (3H, d, J=6.8Hz), 1.20-1.42 (19H, m), 1.45-1.98 (15H, m), 2.20 (3H, s), 2.57-2.79(6H, m), 2.85-3.00 (1H, m), 3.03-3.17 (1H, m), 3.30-3.57 (3H, m),3.72-3.83 (5H, m), 3.96-4.00 (1H, m), 4.22 (1H, d, J=8.8 Hz), 4.29-4.35(3H, m), 4.95 (1H, dd, J=10.4, 2.4 Hz), 7.22-7.45 (17H, m), 7.71 (1H,s), 7.78 (2H, d, J=8.4 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 10.7, 11.1, 13.1,14.6, 16.6, 19.3, 19.9, 21.4, 22.3, 26.3, 29.6, 30.2, 36.5, 37.0, 38.8,42.5, 43.0, 48.3, 49.3, 49.7, 50.4, 51.4, 53.7, 57.7, 60.1, 65.5, 69.7,70.5, 76.7, 78.7, 79.4, 81.8, 92.8, 104.1, 119.7, 126.0, 128.4, 129.2,129.4, 130.0, 132.3, 138.9, 141.3, 147.6, 156.3, 169.8, 177.6, 182.5,204.5; HRMS (ESI) calc for [C67H87N7O11+H]+ 1166.6536. found 1166.6478

Tricyclic Ketolide-N-benzyltriazolylocta-N-Otrityl (11c)

Reaction of 4′-ethynylbenzyl tricyclic ketolide 9 (0.075 g, 0.102 mmol)and 6-azidoocta-o-trityl hydroxamic acid 10c (0.045 g, 0.102 mmol) inanhydrous THF (5 ml) within 22 h as described for the synthesis andpurification of 11a gave 94 mg (79%) of 11c as a yellowish solid. ¹H NMR(CDCl₃, 400 MHz) δ 0.83 (3H, t, J=7.2 Hz), 1.04 (3H, d, J=7.2 Hz),1.15-1.41 (21H, m), 1.45-1.95 (15H, m), 2.18 (3H, s), 2.55-2.73 (6H, m),2.87-2.97 (1H, m), 3.03-3.10 (1H, m), 3.28-3.57 (3H, m), 3.70-3.82 (5H,m), 3.93-3.97 (1H, m), 4.21 (1H, d, J=8.4 Hz), 4.27-4.34 (3H, m), 4.94(1H, dd, J=12.8, 2.4 Hz), 7.25-7.42 (17H, m), 7.71 (1H, s), 7.76 (2H, d,J=8.0 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 10.7, 11.1, 13.1, 14.6, 16.6,19.3, 19.9, 21.4, 22.3, 26.4, 28.8, 29.6, 30.4, 36.6, 37.0, 38.8, 42.5,42.9, 48.3, 49.3, 49.7, 50.6, 51.4, 53.7, 57.7, 60.1, 65.5, 69.8, 70.5,76.7, 78.7, 79.4, 81.8, 93.8, 104.1, 119.6, 126.0, 127.7, 128.3, 128.8,129.2, 129.4, 130.0, 132.7, 138.9, 141.3, 147.7, 156.3, 169.8, 177.0,182.4, 204.4; HRMS (ESI) calc for [C68H89N7O11+H]+ 1180.6692. found1180.6637

Tricyclic Ketolide-N-benzyltriazolylnona-N-Otrityl (11d)

Reaction of 4′-ethynylbenzyl tricyclic ketolide 9 (0.075 g, 0.102 mmol)and 6-azidonona-o-trityl hydroxamic acid 10d (0.046 g, 0.102 mmol) inanhydrous THF (5 ml) within 20 h as described for the synthesis andpurification of 11a gave 93 mg (76%) of 11d as a yellowish solid. ¹H NMR(CDCl₃, 400 MHz) δ 0.85 (3H, t, J=8.0 Hz), 1.05 (3H, d, J=6.8 Hz),1.16-1.44 (23H, m), 1.45-1.96 (15H, m), 2.20 (3H, m), 2.57-2.78 (6H, m),2.90-2.99 (1H, m), 3.02-3.12 (1H, m), 3.30-3.57 (3H, m), 3.72-3.84 (5H,m), 3.96-4.00 (1H, m), 4.29 (1H, d, J=8.4 Hz), 4.31 (1H, d, J=7.2 Hz),4.37 (2H, J=7.2 Hz), 4.95 (1H, dd, J=12.8, 2.4 Hz), 7.19-7.45 (17H, m),7.72 (1H, s), 7.78 (2H, d, J=8.0 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 10.7,11.1, 13.1, 14.6, 16.6, 19.3, 19.9, 21.4, 22.3, 26.6, 28.9, 29.2, 29.6,29.9, 30.5, 36.5, 37.0, 37.1, 38.8, 42.5, 43.0, 48.3, 49.3, 49.7, 50.6,51.4, 57.7, 60.1, 65.5, 65.6, 69.7, 70.5, 76.7, 78.7, 79.4, 81.8, 94.3,104.1, 119.6, 126.0, 128.3, 128.8, 129.0, 129.2, 129.4, 130.0, 132.3,138.9, 141.3, 147.6, 156.3, 169.8, 178.0, 181.5, 204.5; HRMS (ESI) calcfor [C69H91N7O11+H]+ 1194.6849. found 1194.6838

Tricyclic Ketolide-N-benzyltriazolyldeca-N-Otrityl (11e)

Reaction of 4′-ethynylbenzyl tricyclic ketolide 9 (0.075 g, 0.102 mmol)and 6-azidodeca-o-trityl hydroxamic acid 10e (0.048 g, 0.102 mmol) inanhydrous THF (5 ml) within 20 h as described for the synthesis andpurification of 11a gave 98 mg (80%) of 11e as a yellowish solid. ¹H NMR(CDCl₃, 400 MHz) δ 0.83 (3H, t, J=6.8 Hz), 1.03 (3H, d, J=7.2 Hz),1.15-1.40 (25H, m), 1.44-1.96 (15H, m), 2.17 (3H, s), 2.57-2.78 (6H, m),2.87-2.95 (1H, m), 3.03-3.08 (1H, m), 3.28-3.53 (3H, m), 3.70-3.82 (5H,m), 3.92-3.96 (1H, m), 4.20 (1H, d, J=8.4 Hz), 4.29 (1H, d, J=6.8 Hz),4.33 (2H, t, J=7.2 Hz), 4.93 (1H, d, J=10.0 Hz), 7.26-7.43 (17H, m),7.73 (1H, s), 7.76 (2H, d, J=8.0 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 10.7,11.1, 13.1, 14.6, 16.6, 19.3, 19.8, 21.4, 22.3, 26.6, 29.1, 29.3, 29.6,29.9, 30.5, 36.6, 37.0, 38.8, 42.5, 43.0, 48.3, 49.3, 49.7, 50.6, 51.4,57.7, 60.1, 65.5, 69.7, 70.5, 76.7, 78.7, 79.4, 81.8, 93.4, 104.1,199.6, 126.0, 128.3, 129.2, 129.4, 130.0, 138.9, 147.6, 156.3, 169.8,177.6, 182.0, 204.5; HRMS (ESI) calc for [C70H93N7O11+H]+ 1208.7005.found 1208.6888

Tricyclic Ketolide-N-benzyltriazolylhexahydroxamic acid (56a)

Protocol B: To a solution of tricyclicketolide-N-benzyltriazolylhexa-N-Otrityl 11a (0.120 g, 0.105 mmol) inmethylene chloride (1 ml) at 0° C., was added thioanisole (0.2 ml) andTFA (0.2 ml) dropwise. Stirring was continued at 0° C. for 2 h afterwhich TLC analysis indicated completion of reaction. Excess TFA andsolvent were evaporated off and immediately placed back in the ice-bathfollowed by addition of PBS buffer (10 ml). Saturated NaHCO₃ was addeddropwise until the pH was neutral. Extraction with 20% MeOH/CH₂Cl₂ (10ml×3), drying over Na₂SO₄, and evaporation of solvent afforded liquidcrude product which was then purified by prep TLC (silica, 12:1:0.1CH₂Cl₂/MeOH/conc. NH₄OH) to afford 47 mg (50%) of 56a as a yellowishsolid.

Protocol C: To a mixture of tricyclicketolide-N-benzyltriazolylhexa-N-Otrityl 11a (0.136 g, 0.118 mmol) inCH₂Cl₂/MeOH (2 ml: 2 ml) was added BF₃.OEt₂ (0.033 g, 0.236 mmol) andstirred at room temperature for 45 min. Dilute NaHCO₃ (20 ml) was addeduntil pH=8, and then extracted with 10% MeOH/CH₂Cl₂ (10 ml×4), driedover Na₂SO₄ and concentrated in vacuo. The crude product was purified byprep TLC (silica, 12:1:0.1 CH₂Cl₂/MeOH/conc. NH₄OH) to give 64 mg (60%)of 56a as a yellowish solid.

Protocol D: 4′-Ethynylbenzyl tricyclic ketolide 9 (0.050 g, 0.065 mmol)and 6-azidohexahydroxamic acid 13a (0.025 g, 0.145 mmol) were dissolvedin anhydrous THF (7 mL) and stirred under argon at room temperature.Copper (I) iodide (0.007 g, 0.036 mmol) and Hunig's base (0.5 mL) wereadded to the reaction mixture, and stirring continued for 12 h. Thereaction mixture was diluted with 10% MeOH/CH2Cl2 (20 mL) and washedwith 1:4 NH4OH/saturated NH4Cl (3×10 mL) and saturated NH4Cl (10 mL).The organic layer was dried over Na2SO4 and concentrated in vacuo. Thecrude product was purified by prep TLC (silica, 12:1:0.1CH2Cl2/MeOH/conc. NH4OH) to give 20 mg (34%) of 56a as a brown solid.

¹H NMR (CDCl₃, 400 MHz) δ 0.85 (3H, t, J=8.0 Hz), 1.05 (3H, d, J=7.2Hz), 1.20-2.00 (30H, m), 2.13-2.18 (2H, m), 2.20 (3H, s), 2.58-2.72 (6H,m), 2.92-2.98 (1H, m), 3.05-3.09 (1H, m), 3.30-3.55 (3H, m), 3.70-3.84(5H, m), 3.95-3.98 (1H, m), 4.22 (1H, d, J=8.0 Hz), 4.31-4.38 (3H, m),4.95 (1H, d, J=8.8 Hz), 7.33 (2H, d, J=8.0 Hz), 7.77 (3H, m); ¹³C NMR(CDCl₃, 100 MHz) δ 10.6, 11.1, 13.1, 14.6, 16.5, 19.3, 19.8, 21.4, 22.3,24.7, 25.9, 29.7, 29.9, 30.0, 36.6, 37.1, 38.8, 42.5, 42.8, 49.4, 49.6,50.3, 51.4, 53.7, 57.7, 60.1, 65.5, 69.7, 70.5, 76.7, 78.7, 79.3, 81.8,104.1, 120.1, 126.0, 129.5, 129.7, 139.1, 147.7, 156.3, 169.8, 171.1,182.3, 204.5; HRMS (EST) talc for [C47H71N7O11+H]+ 910.5284. found910.5279; Melting point 127-130° C.

Tricyclic Ketolide-N-benzyltriazolylheptahydroxamic acid (56b)

According to protocol B, reaction of 11b (0.132 g, 0.113 mmol) in CH₂Cl₂(1 ml), thioanisole (0.3 ml) and TFA (0.3 ml) for 3 h afforded 56 mg(55%) of 56b as a yellowish solid.

According to protocol C, reaction of 11b (0.15 g, 0.127 mmol) andBF₃.OEt₂ (0.037 g, 0.257 mmol) afforded 65 mg (55%) of 56b as ayellowish solid.

Reaction of 4′-ethynylbenzyl tricyclic ketolide 9 (0.048 g, 0.063 mmol)and 7-azidoheptahydroxamic acid 13b (0.020 g, 0.107 mmol) within 12 h,according to the protocol D described for the synthesis of compound 56a,gave 22 mg (38%) of 56b as a brown solid.

¹H NMR (CDCl₃, 400 MHz) δ 0.83 (3H, t, J=7.6 Hz), 1.03 (3H, d, J=6.4Hz), 1.18-1.90 (32H, m), 2.10-2.13 (2H, m), 2.17 (3H, s), 2.58-2.73 (6H,m), 2.93-2.98 (1H, m), 3.03-3.07 (1H, m), 3.28-3.60 (3H, m), 3.70-3.81(5H, m), 3.98 (1H, m), 4.20 (1H, d, J=8.4 Hz), 4.29-4.38 (3H, m), 4.93(1H, d, J=10.4 Hz), 7.30 (2H, d, J=7.6 Hz), 7.75 (2H, d, J=7.6 Hz), 7.78(1H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 10.6, 11.1, 13.1, 14.6, 16.5, 19.3,19.8, 21.4, 22.3, 25.2, 26.0, 28.2, 29.7, 29.9, 30.1, 36.6, 37.0, 38.8,42.5, 42.7, 48.2, 49.3, 49.5, 50.4, 51.4, 53.7, 57.7, 60.1, 65.5, 69.7,70.5, 76.7, 78.7, 79.3, 81.8, 104.0, 120.0, 126.0, 129.4, 129.8, 139.1,147.7, 156.3, 169.8, 171.2, 182.4, 204.5; HRMS (ESI) calc for[C48H73N7O11+H]+ 924.5440. found 924.5422; Melting point 128-131° C.

Tricyclic Ketolide-N-benzyltriazolyloctahydroxamic acid (56c)

Reaction of 11c (0.092 mg, 0.079 mmol) in CH₂Cl₂ (1 ml), thioanisole(0.2 ml) and TFA (0.2 ml) according to protocol B afforded 37 mg (51%)of 56c as a yellowish solid.

¹H NMR (CDCl₃, 400 MHz) δ 0.83 (3H, t, J=6.4 Hz), 1.03 (3H, d, J=5.6Hz), 1.18-1.60 (21H, m), 1.66-2.1 (15H, m), 2.18 (3H, s), 2.56-2.70 (6H,m), 290-2.98 (1H, m), 3.02-3.07 (1H, m), 3.28-3.58 (3H, m), 3.70-3.81(5H, m), 3.94-3.97 (1H, m), 4.20 (1H, d, J=7.2 Hz), 4.28 (1H, d, J=6.0Hz), 4.35 (2H, m), 4.93 (1H, d, J=10.4 Hz), 7.30 (2H, d, J=7.2 Hz),7.73-7.76 (3H, m); ¹³C NMR (CDCl₃, 100 MHz) δ 10.6, 11.1, 13.1, 14.4,14.6, 16.5, 19.3, 19.8, 21.3, 21.4, 22.3, 25.2, 26.1, 28.3, 29.7, 29.9,30.2, 32.9, 36.6, 37.1, 38.8, 42.5, 42.7, 48.2, 49.4, 49.5, 50.5, 51.4,57.7, 60.1, 60.6, 65.5, 69.7, 70.5, 76.7, 78.7, 79.3, 81.8, 104.0,119.9, 126.0, 129.5, 129.8, 139.0, 147.7, 156.3, 169.8, 171.7, 182.4,204.5; HRMS (ESI) calc for [C49H75N7O11+H]+ 938.5597. found 938.5556;Melting point 113-115° C.

Tricyclic Ketolide-N-benzyltriazolylnonahydroxamic acid (56d)

Reaction of 11d (0.091 mg, 0.077 mmol) in CH₂Cl₂ (1 ml), thioanisole(0.2 ml) and TFA (0.2 ml) according to protocol B afforded 43 mg (60%)of 56d as a yellowish solid.

¹H NMR (CDCl₃, 400 MHz) δ 0.83 (3H, t, J=7.2 Hz), 1.03 (3H, d, J=7.2Hz), 1.17-1.60 (23H, m), 1.66-2.14 (15H, m), 2.17 (3H, s), 2.58-2.73(6H, m), 2.87-2.95 (1H, m), 3.00-3.07 (1H, m), 3.27-3.55 (3H, m),3.70-3.82 (5H, m), 3.93-3.97 (1H, m), 4.20 (1H, d, J=8.8 Hz), 4.28 (1H,d, 7.2 Hz), 4.35 (2H, t, J=6.8 Hz), 4.92 (1H, dd, J=10.0, 1.2 Hz), 7.30(2H, d, J=8.0 Hz), 7.75 (3H, m); ¹³C NMR (CDCl₃, 100 MHz) δ 10.7, 11.1,13.1, 14.6, 16.5, 19.3, 19.8, 21.4, 22.3, 25.4, 26.3, 28.6, 28.8, 28.9,29.6, 29.9, 30.3, 32.9, 36.6, 37.1, 38.8, 42.5, 42.8, 48.2, 49.3, 49.6,50.6, 51.4, 57.7, 60.1, 65.5, 69.7, 70.5, 76.7, 78.7, 79.3, 81.8, 104.1,119.8, 126.0, 129.5, 129.8, 139.0, 147.7, 156.3, 169.8, 171.3, 182.9,204.5; HRMS (ESI) calc for [C50H77N7O11+H]+ 952.5753. found 952.5728;Melting point 112-115° C.

Tricyclic Ketolide-N-benzyltriazolyldecahydroxamic acid (56e)

Reaction of 11e (0.097 mg, 0.081 mmol) in CH₂Cl₂ (1 ml), thioanisole(0.2 ml) and TFA (0.2 ml) according to protocol B afforded 42 mg (55%)of 56e as a yellowish solid.

¹H NMR (CDCl₃, 400 MHz) δ 0.83 (3H, t, J=7.2 Hz), 1.03 (3H, d, J=6.4Hz), 1.18-1.61 (25H, m), 1.66-2.14 (15H, m), 2.18 (3H, s), 2.59-2.73(6H, m), 2.89-2.94 (1H, m), 3.01-3.07 (1H, m), 3.28-3.54 (3H, m),3.70-8.83 (5H, m), 3.93-3.97 (1H, m), 4.20 (1H, d, J=8.4 Hz), 4.28 (1H,d, J=6.8 Hz), 4.36 (2H, t, J=7.2 Hz), 4.92 (1H, dd, J=10.4, 1.6 Hz),7.30 (2H, d, J=8.0 Hz), 7.76 (3H, m); ¹³C NMR (CDCl₃, 100 MHz) δ 10.7,11.1, 13.1, 14.4, 14.6, 16.5, 19.3, 19.9, 21.4, 22.3, 25.5, 26.3, 28.7,28.9, 29.0, 29.6, 29.9, 30.3, 33.1, 36.6, 37.1, 38.8, 42.5, 42.7, 48.2,49.4, 49.5, 50.6, 51.4, 57.7, 60.1, 60.6, 65.5, 69.7, 70.5, 76.7, 78.7,79.3, 81.8, 104.0, 119.8, 126.0, 129.5, 129.8, 139.0, 147.7, 156.3,169.8, 171.7, 182.9, 204.5; HRMS (ESI) calc for [C51H79N7O11+H]+966.5910. found 966.5919; Melting point 123-126° C.

Example 5 Anti-HDAC Activity of Nonpeptide Macrocyclic HDAC Inhibitors

Inhibition of HeLa nuclear extract HDAC1/2 and HDAC8 by compounds 7-14,23-30, 36, and 38 was evaluated in a Fluor de Lys assay according to themanufacture's protocol. Each IC₅₀ value was obtained by averaging threeindependent experiments. This data is shown in Table 4. The compoundsdisplayed both linker-length and macrolide-type dependent HDACinhibition activities with IC₅₀ in low nanomolar range.

TABLE 4 Inhibitory activity of HDAC inhibitors Compound HDAC ½ (nM) HDAC8 (nM) 7 91.6 4,730 8 88.8 3,740 9 13.85 994 10 10.56 1,020 11 58.887,130 12 72.44 6,780 13 145.50 11,050 14 226.73 N.D. 23 36.98 3,990 2444.26 4,750 25 4.09 1,890 26 1.87 1,390 27 55.59 5,880 28 123.03 4,42029 169.80 10,550 30 223.36 N.D 36 107.1 6,680 38 109.8 2,320

The ketolides 56 were also evaluated for their HDAC inhibitory activityagainst HDACs 1 and 2 from HeLa cell nuclear extract, HDAC6, and HDAC8using the Fluor de Lys assay. The results are shown in Table 5.

TABLE 5 HDAC inhibition activity (IC₅₀) and Isoform Selectivity oftricyclic ketolide-based HDAC inhibitors HDAC8: HDAC6: Nuclear NuclearNuclear Extract Extract Extract HDAC Isoform HDAC Isoform Compd n (nM) 8(nM) Selectivity 6 (nM) Selectivity 56a 1 7.77 796.2 102.5 1180.1 151.956b 2 1.03 544.6 528.7 728.7 707.5 56c 3 104.2 1909.3 18.3 1709.8 16.456d 4 163.6 2859.9 17.5 1916.9 11.7 56e 5 208.2 4557.8 21.9 3203.1 15.4

Results show a linker-dependent anti-HDAC activity which peaked withcompound 56b, an analog having 6 methylene spacers separating thetriazole ring from the zinc binding hydroxamic acid group (Table 5).Compound 56b potently inhibits the deacetylase activity of HDACs 1 and 2with a single digit nanomolar IC₅₀. Compounds 56c-e, analogs havinglonger methylene spacers than those of 56b, show a progressive reductionin anti-HDAC activity with increase in methylene spacer length. The HDACinhibition profiles of these triketolide-derived HDACi paralleled thosewe previously reported for the macrolide-derived HDACi.

To obtain evidence for the HDAC isoform selectivity, the triketolideHDAC inhibitors were tested against HDAC6 and HDAC8. Compared to SAHA,compounds 56a and 56b are more selective for HDAC1/2 with selectivityindices comparable to those of their 14-membered macrolide congeners.Compounds 56c-e are less selective for either HDAC isoform compared to56a-b. However, 56c-e have improved HDAC6 and comparable HDAC8 isoformselectivity relative to SAHA (Table 5).

Example 6 Evaluating in Vitro Anti-Cancer Activity of HDAC Inhibitors

The potency of compounds in Table 5 were investigated by determining thedrug concentrations necessary for 50% inhibition of cell viability(IC₅₀) in SKMES 1, NCI-1169, DU 145 cells, lung fibroblasts, and HMEC.Drug concentrations necessary for 50% inhibition of cell viability(EC₅₀) were quantitatively measured using trypan blue exclusionaccording to literature protocol (Mosmann, T. (1983). J. Immunol.Methods 65: 55; Chen et al. (2008) Bioorg. Med. Chem. 16: 4839). Table 6shows the EC₅₀ values for each compound. All compounds inhibit theproliferation of the transformed cells studied with EC₅₀ in lowmicromolar range. Most importantly, these compounds are less toxic tountransformed cell-lines (lung fibroblast and HMEC) that we have studiedto date.

TABLE 6 Cell growth inhibitory data Lung SKMES 1 NCI-H69 DU-145fibroblast HMEC Compound (uM) (uM) (uM) (uM) (uM) 7 1.79 1.921.45 >10 >10 8 1.68 1.77 1.24 >10 >10 9 2.33 3.45 1.88 >10 >10 10 2.563.01 1.97 >10 >10 11 4.89 4.56 5.89 >10 >10 12 4.67 3.99 5.68 >10 >10 137.54 8.45 >10 >10 >10 23 2.15 2.67 2.98 >10 >10 24 1.95 1.923.29 >10 >10 25 1.33 1.45 1.12 >10 >10 26 1.28 1.49 1.05 >10 >10 27 4.895.67 6.97 >10 >10 28 4.45 5.09 5.78 >10 >10 29 7.12 7.29 8.14 >10 >10

The effect of compounds 56a-e on cancer cell lines: prostate (DU-145),lung (A549), and breast cancer (MCF-7) and a non-transformed cell, humanlung fibroblast cell (Hs1.Lu) were also evaluated. Drug concentrationsnecessary for 50% inhibition of cell viability (EC₅₀) werequantitatively measured using MTS colorimetric assay as described above.Table 7 shows the EC₅₀ values of each compound.

TABLE 7 Anti-proliferative activity (μM) of tricyclic ketolide-basedHDAC inhibitors DU-145 A549 MCF-7 Hs1.Lu Normal Lung Compd n (μM) (μM)(μM) Fibroblast (μM) 56a 1 1.64 1.17 1.26 N.D. 56b 2 0.82 0.66 0.75 N.D.56c 3 3.70 1.64 1.35 N.D. 56d 4 3.89 2.48 2.56 N.D. 56e 5 4.83 2.81 3.68N.D.

Compounds 56a-e inhibit the proliferation of all transformed cellsstudied with anti-proliferative activity that closely matched their invitro ant-HDAC activity. Specifically, compound 56b has the most potentanti-proliferative activity with high nanomolar efficacy against allthree cancer cell lines. Moreover, none of the compound shows anydiscernible toxicity against normal Hs1.Lu cells at drug concentrationsin excess of 10 μM (Table 7). These data showed that the triketolidehydroxamates reported here are selectively toxic to the transformedcells.

Example 7 Antiparasitic Activity of Non-Peptide Macrocyclic HistoneDeacetylase Inhibitors

Macrocyclic histone deacetylase inhibitors were evaluated for parasiticactivity against Plasmodium falciparum and Leismania donovani. P.falciparum and L. donovani are the causative parasites of malaria andleishmaniasis, two human diseases which constitute a serious threat topublic health in tropical and sub-tropical countries. Antimalarialactivity was evaluated in vitro using chloroquine-sensitive (D6, SierraLeone) and chloroquine-resistant (W2, Indochina) strains of P.falciparum. Antileishmanial activity was evaluated in vitro against thepromastigote stage of L. donovani.

Plasmodium growth inhibition was determined by a parasite lactatedehydrogenase assay using Malstat reagent. Inhibition of viability ofthe promastigote stage of L. donovani was determined using standardAlamar blue assay, modified to a fluorometric assay. Amphotericin B andpentamidine, standard antileishmanial agents; chloroquine andartimisinin, standard antimalarial; and suberoylanilide hydroxamic acid(SAHA), standard HDACi were all used as positive controls. To determineselective toxicity index, all compounds were tested againstnontransformed mammalian cell lines namely, monkey kidney fibroblasts(Vero) and murine macrophages (J774.1) using Neutral Red assay. Theresults are shown in Table 8.

TABLE 8 Antiparasitic activity of non-peptide HDAC inhibitors HDACAntimalarial Activity Cyto- Inhibi- Antileshmanial Plasmodium Plasmodiumtoxicity tion activity falciparum falciparum (VERO) S.I. IC₅₀ IC₅₀ IC₉₀(D6 clone) (W2 clone) IC₅₀ D6 Compd R n (nM)^(a) (μg/ml) (μg/ml) IC₅₀(μg/ml) IC₅₀ (μg/ml) (μg/ml) (W2) 1

5 37.0 NA NA 0.90 0.93 NC >5.3 (>5.1) 2

5 44.3 NA NA 1.20 1.40 NC >4.0 (>3.4) 3

5 91.6 NA NA 1.20 1.60 NC >4.0 (>3.0) 4

5 88.8 NA NA 1.30 1.70 NC >3.7 (>2.8) 5

6 4.1 18 NA 0.27 0.31 NC >17.6 (>15.4) 6

6 1.9 NA NA 0.18 0.25 NC >26.4 (>19.0 7

6 13.9 NA NA 0.23 0.10 NC >20.7 (>47.6) 8

6 10.6 NA NA 0.16 0.17 NC >29.8 (>28.0 9

7 55.6 20 34 2.50 2.00 NC >1.9 (>2.4) 10

7 123.0 NA NA 3.00 2.70 NC >1.6 (>1.8) 11

7 58.9 NA NA 2.40 2.30 NC >2.0 (>2.1) 12

7 72.4 NA NA 3.50 3,20 NC >1.4 (>1.5) 13

8 169.8 3.40 7.00 1.70 1.00 NC >2.8 (>4.8) 14

8 145.5 20 35 1.10 0.83 NC >4.3 (>5.7) 15

9 223.4 3.40 7.00 1.40 0.80 NC >3.4 (6.0) 16

9 226.7 3.50 7.00 0.84 0.88 NC >5.7 (>5.4) Chloroquine — NT NT NT 0.0170.125 NT NT Artetnisinin — NT NT NT 0.004 0.006 NT NT Pentamidine — NT0.90 1.70 NT NT NT NT Amphotericine B — NT 0.18 0.32 NT NT NT NT SAHA —65 22 50 0.25 0.47 1.20 4.8 (2.5) NA = Not active up to the highestconcentration tested NC = Not cytotoxic up to the highest concentrationtested NT = Not tested

The non-peptide macrocyclic HDAC inhibitors potently inhibited theproliferation of both the sensitive and resistant strains of P.falciparum with IC50 ranging from 0.1 μg/mL to 3.5 μg/mL (Table 1). Inparticular, compounds 5-8 in Table 8, derived from either the 14- or15-membered macrolide analogs macrolides and having 6 methylene spacersseparating the triazole ring from the zinc-binding hydroxamic acid group(n=6), have the most potent antimalarial activities in this series.These compounds are equipotent or >4-fold more potent than the controlcompound SAHA. Moreover, they are several folds more selectively toxicto either strains of P. falciparum compared to SAHA. The antimalarialactivities of these macrocyclic HDACi followed a similar trend as theiranti-HDAC activity against HDAC1/2 from HeLa nuclear extract, suggestingthat parasite HDACs could be an intracellular target of the thesecompounds.

Compounds 5 and 9 exhibited modest activity against the promastigotestage of L. donovani. This result is contrary to previous data on simplearyltriazolylhydroxamates which have antimalarial and antileishmanialactivities that followed a similar trend.

A comparison of the antileishmanial activities of compounds 13 and 14,analogs with n=8, revealed an interesting disparity between the activityof 14- and 15-membered macrocyclic rings. 14-Membered compound 13 is 5-to 6-fold more potent than its 15-membered congener 14. However, thisdisparity dissipates after a single methylene group extension (n=9), ascompounds 15 and 16 have virtually indistinguishable antileishmanialactivities. Comparatively, compounds 13, 15 and 16, analogs with themost potent antileishmanial activities, are about 7- to 10-fold morepotent than SAHA and approximately 3-fold less potent than pentamidine.

Since these non-peptide macrocyclic HDACi have nanomolar anti-HDACactivities, the observed disparity in the trend of their antimalarialand antileishmanial activities may have implication in the organizationof the active sites of the relevant P. falciparum and L. donovani HDACisozymes. These observations provide additional evidence of thesuitability of HDAC inhibition as a viable therapeutic option to curbinfections caused by apicomplexan protozoans and trypanosomatids (1, 5,6, 14) and could facilitate the identification of other HDACi that aremore selective for either parasite.

The antiparasitic activity of ketolides 56 were also evaluated againstP. falciparum and L. donovani. The results are shown in Table 9.

TABLE 9 Antiparasitic activity of ketolide non-peptide HDAC inhibitorsAntimalarial Activity Antilieshmanial Plasmodium Plasmodium CytotoxicityActivity falciparum falciparum (VERO) S.I. IC₅₀ IC₉₀ (D6 clone) (W2clone) IC₅₀ D6 Compound n (μg/ml) (μg/ml) IC₅₀ (μg/ml) IC₅₀ (μg/ml)(μg/ml)^(a) (W2) 56a 1 NA NA 0.96 1.80 NC >5.0 (no value) 56b 2 NA NA0.14 0.12 NC  >34 (>39.7) 56c 3 19 36 1.20 1.50 NC >4.0 (>3.2) 56d 4 1432 1.20 1.30 NC >4.0 (>3.7) 56e 5 4.8 23 1.20 0.84 NC >4.0 (>5.7)Chloroquine — NT NT 0.011 0.14 NT NT Artemisinin — NT NT 0.006 0.009 NTNT Pentamidine — 1.4 6.0 NT NT NT NT Amphotericine — 0.08 0.3 NT NT NTNT B SAHA — 22 50 0.25 0.47 1.20   4.8 (2.5)

Compounds 56a-e potently inhibit the proliferation of both the sensitiveand resistant strains of P. falciparum with IC₅₀ ranging from 0.12 μg/mLto 1.5 μg/mL (Table 9). 56b has the most potent antimalarial activitywhich is between 2- to 4-fold more potent than the control compoundSAHA. Moreover, 56b is several folds more selectively toxic to eitherstrains of P. falciparum compared to SAHA. Compounds 56c-e have amoderate to good antileishmanial activity against the promastigote stageof L. donovani activity which peaks with 56e, the analog with thelongest methylene spacers in the series. This result is in contrast tothe antimalarial activity that mirrors compound anti-HDAC activity andit may have implications in the organization of the active sites of therelevant P. falciparum and L. donovani HDAC isozymes. We have observed asimilar methylene spacer-length dependence in the antileishmanialactivity of macrocyclic HDACi 14- and 15-membered macrolides as shown inTable 8. The foregoing observations further support the suitability ofHDAC inhibition as a viable therapeutic strategy to curb infectionscaused by apicomplexan protozoans and trypanosomatids.

Example 8 3-Hydroxypyridine-2-thiones as Zinc Binding Groups for HDACInhibition In Silico Analysis of Pyridinone Zinc Binding Groups

To evaluate the feasibility of pyridinone fragments in HDACi design,fragments were docked against HDAC crystal structures using validatedmolecular dock program (AutoDock 4.2). Three representative pyridinonefragments—3-hydroxypyridine-2-thione (3-HPT),3-hydroxypyridine-4-thiones, and 1-hydroxypyridine-2-thione, thestructures of which are shown below, were docked against HDACmacromolecules. It is noted that the N-1 position of3-hydroxypyridine-2-thione and 3-hydroxypyridine-4-thione could bemodified, as required, to include a prototypical HDACi linker regioncoupled to a surface recognition cap group. Small fragments tend to havelower binding energy and non-specific interaction at the surface. Theseproblems were addressed by: i) selecting compact grid size around theZn²⁺ active site; and ii) selecting thione analogs of pyridinonefragments which are expected to bind more tightly with Zn²⁺ metal ionthan their carbonyl counterparts, thereby improving binding energy.

These fragments were docked against class I HDAC enzymes HDAC1 and HDAC8and structurally distinct HDAC6 which belongs to class II. Theseisoforms have distinct structural differences at the Zn²⁺ active site.Any evidence of isoform selectivity against any one of them shouldestablish a broader isoform profile. The HDAC8 crystal structure (PDBcode: 1VKG) has been solved, while HDAC1 and HDAC6 homology model werebuilt from human HDAC2 (PDB code: 3MAX) and HDAC8 (PDB code: 3FOR) X-raystructure coordinates respectively. These were used to obtain in silicodata for fragment interaction at the active sites. Preliminary dockinganalysis against HDAC isoforms revealed that 3-hydroxypyridine-2-thione(3-HPT), 3-hydroxypyridine-4-thiones, and 1-hydroxypyridine-2-thionebind to the vicinity of Zn²⁺ ion at the active sites of HDAC1, HDAC6 andHDAC8. This suggests that pyridinones can serve as effective ZBGs inHDACi design.

Closer analyses of the docked structures revealed that the orientationof N-1 position of 3-hydroxypyridine-4-thione is pointing towards thebase of the active site pocket instead of pointing toward the surface asseen with 3-HPT. In order to evaluate ZBGs other than hydroxamic acid,3-hydroxypyridine-2-ones were selected for further investigation.

In vitro analysis of 3-hydroxypyridine-2-ones as ZBGs

To verify the in silico findings, an HDAC inhibition assay using3-hydroxypyridine-2-ones was performed to determine their effect onenzyme activity. HDAC inhibition was assayed initially using cell freeassay (Fluor de Lys) as described above.

There is precedence that fluorescently labeled substrates can perturbenzyme activity and the effect of inhibitors. Indeed, many recommendedFluor de Lys substrates have been shown not to be active toward theircorresponding enzymes, including HDAC8, in the absence of fluorophore.Additionally, 3-hydroxypyridine-2-ones are highly colored solids andmight lead to false positive signals. Mindful of these facts, alabel-free SAMDI (self-assembled monolayers for MALDI) mass spectrometryassay was also used to assess HDAC inhibition.

The SAMDI technique, wherein self-assembled monolayers (SAM) ofalkanethiolates on gold are analyzed by matrix assisted laserdesorption/ionization (MALDI) mass spectrometry, has been widely used tocharacterize many biochemical activities, including the deacetylaseenzyme family. The SAMDI assay was used to measure the inhibitionconstant (K₁) of fragments against HDAC1, HDAC6 and HDAC8 using apreviously identified preferred HDAC substrate. 3-hydroxypyridine-2-onewas found to be inactive against the three HDAC isoforms. However, thethione derivative (3-HPT) showed low micromolar IC₅₀ values against theHDAC6 and HDAC8 isoform and inactive against HDAC1 (Table 10).

TABLE 10 Activity of 3-hydroxypyridine-2-one and 3-hydroxypyridine-2-thione as determined by SAMDI analysis. IC₅₀ (nM) Compounds HDAC1 HDAC6HDAC8

NI NI NI

NI 681 ± 110 3675 ± 1201 NI — No significant Inhibition (below 20%Inhibition); % inhibitions of the compounds at 10 μM are given if theIC₅₀ was above 10 μM.

Analysis of substituted 3-hydroxypyridine-2-thiones

After promising results which demonstrated the activity of3-hydroxypyridine-2-thione and suggested potential isoform selectivity,3-HPT analogues substituted at the N-1 nitrogen were investigated todetermine if linker substitution at this position can be tolerated.Accordingly, 1-methyl-3-hydroxypyridine-2-thione was docked againstHDAC6 and HDAC8.

Substitution with N-methyl group didn't alter the docked pose of 3-HPTfragment. The amino acid residues surrounding the docked structure of1-methyl-3-hydroxypyridine-2-thiones were analyzed to further refine thedesign of the linker region. The hydrophobic tunnel which joins the ZBGactive site of HDAC6 to the surface consists of amino acid (AA) residuesLeu74, Pro501, Asp567, Gly619, Phe620, Phe680, Pro748, Tyr782.Hydrophobic aromatic Phe620, Phe680, Tyr782 AA residues are in closeproximity to the N-methyl group of docked1-methyl-3-hydroxypyridine-2-thione's structure. Similarly, thehydrophobic tunnel which joins the ZBG active site of HDAC8 to thesurface consists of amino acid (AA) residues Phe152, Phe208, Tyr306,Trp141, His180, Asp267. Hydrophobic aromatic AA residues Phe152, Phe208,Tyr306, Trp141 are in close proximity of N-methyl group of docked1-methyl-3-hydroxypyridine-2-thione's structure. As a consequence,aromatic linkers containing, for example, phenyl, biphenyl or triazolerings, might contribute to pi-stacking interactions with aromatic AA,thereby enhancing binding energy and improve compounds' affinity forHDAC6 and HDAC8.

Compounds 138-140, 3-hydroxypyridine-2-thiones containing aromaticsubstituents attached to the N-1 nitrogen, were investigated in silico.

Analysis of HDAC6 docking with compound 138 reveals hydrophobicinteraction between the compound 138 phenyl ring and AA residues Phe620and Pro501. However, upon addition of a 2^(nd) phenyl ring, as incompound 139, the compound adopts a higher energy confirmation whichloses hydrophobic interaction as seen in compound 138.

In the case of HDAC8 docking, analysis of compounds 138, 139, and 140revealed that the phenyl ring and the triazole group participated inpi-stacking interactions with aromatic AA Trp141 in HDAC8. In compound139, containing a biphenyl substitution, the 1^(st) phenyl ring adjacentto ZBG maintained its pi-stacking interaction with Trp141 and the 2^(nd)phenyl ring orients in deep subpocket near the active site, albeitwithout any significant interaction. However, it is interesting to notethat the 2^(nd) phenyl ring of compound 139 is surrounded by amino acidswhich can potentially contribute to a low energy conformation byexploiting hydrogen bonding and hydrophobic interaction surrounding it.Similarly, Pro74 and Leu749 surround the 2^(nd) phenyl ring of compound139 in HDAC6 docked conformation. This suggests that substitution of the2^(nd) phenyl ring with hydrogen bonding or hydrophobic groups willpotentially lower binding energy and improve potency.

To investigate the effect of incorporation of functional groups on the2^(nd) phenyl group of the 3-hydroxypyridine-2-thiones, compoundspossessing varied substituents on the second phenyl ring were evaluated.The proposed modifications allow the 3-hydroxypyridine-2-thiones toparticipate in hydrogen bonding as well as hydrophobic interactions withAA residues surrounding the 2^(nd) phenyl group of 139. The compoundschosen for initial investigation are shown below

Initial fragments 142a-142c and 144a-144c were synthesized in 2 to 3steps as indicated in Scheme 17. Synthesis involved coupling of methyliodide and corresponding benzyl bromides with commercially available3-methoxypyridine-2-one to give the O-methyl protected intermediates141a-141c. Thione intermediates were synthesized from their carbonylprecursors using Lawesson's reagent chemistry. Finally, deprotection ofO-methyl group by lewis acid BBr₃ gave the desired products 142a-142cand 144a-144c.

The proposed routes to the biphenyl fragments require crucialintermediate 145 which was obtained by coupling of 4-bromobenzylbromidewith 3-methoxypyridine-2-ones (Scheme 18). Standard Suzuki coupling withcorresponding boronic acids gave intermediates 146a-146i. Similarly,Lawesson's chemistry was used to prepare thione intermediates.Deprotection of the O-methyl group yielded the final biphenyl compounds148a-148i and 149a-149i (Scheme 18).

Synthesis of triazole based compounds 153a-153b and 154a-154b is shownin Scheme 18. It involves the intermediacy of terminal alkyne 150, byN-alkylation reaction of 3-methoxypyridine-2-one with propargyl bromide.Cu(I)-catalyzed Huisgen cycloaddition gave the cyclized product151a-151b. Lawesson's chemistry was again used to gain access tothiolated analogs. Deprotection of O-methyl group was achieved byreaction with BBr₃ (Scheme 19).

The activity of these compounds was evaluated using SAMDI analysis asdescribed above. There is clear distinction in the HDAC inhibitionactivities of the carbonyl and thione analogs with the latter showinghigher potency (Table 11). The enhanced anti-HDAC activity of the thioneanalogs could be attributed to thiophilicity which favors Zn²⁺ binding.Most of the compounds in this series showed more selectivity towardsHDAC6 and HDAC8 compared to HDAC1. A head to head comparison of HDAC6inhibition showed that compound 144b which contains one phenyl ring hasbetter activity than biphenyl compound 144e. Similarly, for HDAC8compound 144b showed 2 fold superior activity when compared to 144e.This observation is consistent with the docking result as the 2^(nd)phenyl ring doesn't take part in any meaningful interactions withresidues on HDAC6 and HDAC8 outer rims. However, replacement of the2^(nd) phenyl ring of compound 144c with a 4-pyridyl ring results inslightly better activity against HDAC8, however this substitution wasn'ttolerated very well against HDAC6 resulting in 4-fold diminishedactivity when compared to 144b.

Substitution of 2^(nd) phenyl ring with electron donatingN,N-dimethylamino group at para position (149g) has similar effect as4-pyridyl substitution (149i) for HDAC8. Importantly, it resulted inloss of activity against HDAC6, making it distinctively selective forHDAC8 (Table 11). On the contrary, combination of pyridine andN,N-dimethylamine substitutions as seen in compound 149h resulted inHDAC6 selectivity as compared to HDAC1 and HDAC8. Incorporation ofmethyl group, another electron donating group, revealed an interestingparadigm of selectivity for HDAC6 and HDAC8. In case of HDAC6,ortho-substitution found to be more favored, followed by meta and thenpara. Conversely, for HDAC8, trend for potency was reversed withpara-substitution showing the best IC₅₀ values (comparing compounds149d, 149e, 149f in Table 11). Ortho-cyano substitution was alsotolerated very well for electron withdrawing cyano group in case ofHDAC6 with IC₅₀ value of 372 nM, followed by para-substitution.Meta-cyano substitution was least tolerated. In case of HDAC8, changingsubstitution pattern didn't change IC₅₀ values significantly showingapproximately 2 μM activity (comparing compounds 149a, 149b, 149c inTable 11). Interestingly, triazole substituted compounds 154a was foundto be more active than corresponding biphenyl compound 144c for HDAC8inhibition. However, a para-N,N-dimethylamino substitution of 154a whichresulted in compound 154b was not beneficial. This is contrary to theeffect of a similar substitution on the biphenyl compound (Table 11,comparing 144c and 149g; 154a and 154b) where para-N,N-dimethylaminosubstitution showed enhancement of activity. In case of HDAC6inhibition, biphenyl compound 144c showed superior activity whencompared to triazole compound 154a which was also not surpassed by apara-N,N-dimethylamino substitution as seen in 154b. This SAR providedselective inhibitors of HDAC6 and HDAC8, albeit with lower micromolaractivity.

TABLE 11 In-vitro HDAC inhibition of 3-hydroxypyridine-2-thiones IC₅₀(nM)* COMPOUNDS HDAC1 HDAC6 HDAC8

  142a NI NI  8%

  142b NI NI  8%

  144b NI 457 ± 27  1272 ± 200 

  142c NI NI  8%

  144c NI 847 ± 188 4283 ± 1548

  148a NI NI 16%

  149a NI 957 ± 159 2075 ± 459 

  148b NI NI  9%

  149b NI 44% 1701 ± 717 

  148c NI  5%  9%

  149c NI 372 ± 35  1907 ± 771 

  148d NI NI 25%

  149d NI 454 ± 42  800 ± 304

  148e NI NI NI

  149e NI 812 ± 286 2496 ± 1180

  148f NI NI  9%

  149f NI 306 ± 69  3105 ± 1649

  148g NI 11% 35%

  149g NI NI 2858 ± 944 

  148h NI NI 37%

  149h NI 2390 ± 458  34%

  148i NI NI 16%

  149i NI 2204 ± 355  2780 ± 323 

  152a NI NI 17%

  154a NI 41% 1570 ± 1067

  152b NI NI  8%

  154b NI 1023 ± 99  1868 ± 723  SAHA 38 +/− 2 95% 232 ± 19  SAHA wassused for a positive control; NI — No significant Inhibition (below 20%Inhibition); % inhibitions of the compounds at 10 μM are given if theIC₅₀ was above 10 μM.

Synthetic Procedures

Bromoalkanoic acid, benzyl bromide, 4-bromobenzylbromide,4-(bromomethyl)-1,1′-biphenyl, 3-methoxy-2(1H)-pyridone, propargylbromide, phenylacetylene and representative boronic acids were purchasedfrom either Sigma-Aldrich or Alfa-Aesar. Anhydrous solvents and otherreagents were purchased and used without further purification. Analtechsilica gel plates (60 F₂₅₄) were used for analytical TLC, and Analtechpreparative TLC plates (UV 254, 2000 μm) were used for purification. UVlight was used to examine the spots. Silica gel (200-400 Mesh) was usedin column chromatography. NMR spectra were recorded on a Varian-Gemini400 magnetic resonance spectrometer. ¹H NMR spectra were recorded inparts per million (ppm) relative to the peak of CDCl₃, (7.24 ppm), CD₃OD(3.31 ppm), or DMSO-d₆ (2.49 ppm). ¹³C spectra were recorded relative tothe central peak of the CDCl₃ triplet (77.0 ppm), CD₃OD (49.0 ppm), orthe DMSO-d₆ septet (39.7 ppm), and were recorded with completeheterodecoupling. Multiplicities are described using the abbreviation s,singlet; d, doublet, t, triplet; q, quartet; m, multiplet; and app,apparent. High-resolution mass spectra were recorded at the GeorgiaInstitute of Technology mass spectrometry facility in Atlanta. Synthesisof 138a was adapted from literature protocol. Azidoalkanols (138a-138f)were prepared according to literature protocols and was used withoutfurther purification. Synthesis of phenyl azide and4-azido-N,N-dimethylaniline were adapted from literature protocols.

Synthesis of 1-methyl-3-methoxypyridine-2-one (141a)

To a stirring reaction mixture of 3-methoxypyridine-2-one (0.2 g, 1.6mmol) and KOH (0.18 g, 3.2 mmol) in methanol was added MeI (0.68 g, 4.8mmol) dropwise in condenser equipped round bottom flask. After overnightstirring at room temperature revealed quantitative formation of product.Reaction mixture was then diluted with water (35 mL) and CHCl₃ (40 mL).Organic layer was washed with water (1×35 mL), brine (1×30 mL) and driedover Na₂SO₄ and solvent was evaporated in vacua to yield pure 141a (0.20g, 89%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 6.66 (m, 1H), 6.35(d, J=7.4 Hz, 1H), 5.83 (t, J=7.1 Hz, 1H), 3.52 (s, 3H), 3.28 (s, 3H).¹³C NMR (100 MHz, CDCl₃) δ 158.01, 149.56, 128.81, 111.91, 104.31,55.44, 37.10.

1-Benzyl-3-methoxypyridine-2-one (141b)

To a stirring reaction mixture of 3-methoxypyridine-2-one (0.2 g, 1.6mmol) and K₂CO₃ (0.66 g, 4.8 mmol) in DMF (8 mL) was added benzylbromide (0.33 g, 1.92 mmol) dropwise in a condenser equipped roundbottom flask. Reaction mixture was then heated to 100° C. Afterovernight stirring, reaction mixture was cooled down and diluted withwater (40 mL) and CHCl₃ (50 mL). Organic layer was washed with water(3×40 mL), brine (1×30 mL) and dried over Na₂SO₄ and solvent wasevaporated in vacuo to yield pure 141b (0.26 g, 75%) as a colorless oilwithout any further purification needed. ¹H NMR (400 MHz, CDCl₃) δ 7.26(m, 5H), 6.85 (dd, J=6.9, 1.7 Hz, 1H), 6.54 (dd, J=7.5, 1.6 Hz, 1H),6.03 (m, 1H), 5.13 (s, 2H), 3.76 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ157.94, 150.05, 136.31, 128.56, 128.01, 127.74, 127.70, 111.82, 104.83,104.81, 55.64, 51.64.

1-(1,1′-Biphenylmethyl)-3-methoxypyridine-2-one (141c)

Reaction of 3-methoxypyridine-2-one (0.2 g, 1.6 mmol), K₂CO₃ (0.66 g,4.8 mmol), (4-bromomethyl)-1,1′-biphenyl (0.47 g, 1.92 mmol) in DMF (8mL) according to method described for synthesis of 141b followed bycolumn chromatography with CH₂Cl₂, acetone (0-15% gradient), MeOH (0-5%gradient) afforded pure 14 k (0.35 g, 76%) as a colorless oil. ¹H NMR(400 MHz, CDCl₃) 7.48 (dd, J=25.9, 20.6 Hz, 4H), 7.32 (m, 5H), 6.88 (m,1H), 6.54 (d, J=7.2 Hz, 1H), 6.04 (t, J=7.1 Hz, 1H), 5.16 (s, 2H), 3.76(s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 157.86, 149.97, 140.51, 140.24,135.28, 128.51, 128.40, 127.71, 127.16, 127.13, 126.74, 111.82, 104.80,55.56, 51.36.

1-Methyl-3-hydroxypyridine-2-one (142a)

To a solution of 141a (0.1 g, 0.68 mmol) in dry CH₂Cl₂ (8 mL) was slowlyadded 1M BBr₃ (0.82 mL) at −30° C. under inert atmosphere. The reactionmixture was stirred for 48 h at room temperature. The mixture was againcooled to −30° C. & then MeOH (5 mL) was slowly added to the mixture.After evaporation of solvent, the residue was adjusted to pH 7 with 1MNaOH and then extracted with CHCl₃ (3×30 mL). The combined organic layerdried over Na₂SO₄. After evaporation of solvent, the residue waspurified by prep-TLC with CH₂Cl₂:Acetone:MeOH (10:1:0.2) to give 61 mg(72%) of pure off white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H),6.78 (dd, J=11.1, 7.1 Hz, 2H), 6.10 (t, J=7.0 Hz, 1H), 3.57 (s, 3H). ¹³CNMR (100 MHz, CDCl₃) δ 158.82, 146.67, 127.63, 114.36, 106.68, 37.28.HRMS (EI) calcd for C₆H₇NO₂ [M]⁺ 125.0477. found 125.0477.

1-Benzyl-3-hydroxypyridine-2-one (142b)

Reaction of 141b (0.15 g, 0.55 mmol) with 1M BBr₃ (0.66 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 142a afforded pure 142b(0.13 g, 90%) as slightly brownish solid. ¹H NMR (400 MHz, CDCl₃) δ7.3.1 (m, 5H), 6.83 (m, 3H), 6.14 (t, J=7.1 Hz, 1H), 5.19 (s, 2H). ¹³CNMR (100 MHz, CDCl₃) δ 158.62, 146.78, 135.82, 128.74, 127.97, 126.53,113.94, 106.95, 52.18. HRMS (EI) calcd for C₁₂H₁₁NO₂ [M]⁺ 201.0792.found 201.0790.

1-(1,1′-Biphenylmethyl)-3-hydroxypyridine-2-one (142c)

Reaction of 14 k (0.13 g, 0.43 mmol) with 1M BBr₃ (0.51 mL) in dryCH₂Cl₂ (7 mL) within 48 h according to procedure 142a afforded pure 142c(0.1 g, 82%) as slightly brownish solid. ¹H NMR (400 MHz, DMSO-d₆) δ9.06 (s, 1H), 7.61 (d, J=7.5 Hz, 4H), 7.36 (m, 6H), 6.70 (d, J=6.5 Hz,1H), 6.12 (t, J=6.9 Hz, 1H), 5.16 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ147.02, 139.77, 139.46, 136.58, 128.93, 128.35, 128.15, 127.49, 126.86,126.66, 114.75, 105.64, 51.07. HRMS (EI) calcd for C₁₈H₁₅NO₂ [M]⁺277.1101. found 277.1103.

1-Benzyl-3-methoxypyridine-2-thione (143b)

A suspension of 141b (0.060 g, 0.28 mmol) and Lawesson's reagent (0.067g, 0.17 mmol) in toluene (10 mL) was heated to reflux overnight.Reaction mixture was then cooled to room temperature. Toluene wasevaporated in vacuo and resulting crude solid was directly loaded onprep-TLC. Elution with CH₂Cl₂:Acetone:MeOH (5:1:0.2) gave 143b 53 mg(82%) of yellow solid.

¹H NMR (400 MHz, CDCl₃) δ 7.30 (m, 6H), 6.65 (d, J=7.7 Hz, 1H), 6.54 (t,J=7.0 Hz, 1H), 5.89 (s, 2H), 3.88 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ173.18, 159.01, 135.18, 131.70, 128.72, 128.03, 127.98, 111.57, 109.63,58.79, 56.61. HRMS (EI) calcd for C₁₃H₁₃NOS [M]⁺ 231.0718. found231.0716.

1-(1,1′-Biphenylmethyl)-3-methoxypyridine-2-thione (143c)

Reaction of 141c (0.13 g, 0.44 mmol) and Lawesson's reagent (0.11 g,0.27 mmol) in toluene according to method described for synthesis of143b afforded 122 mg of 143c (88%) of yellow solid. ¹H NMR (400 MHz,CDCl₃) 7.52 (m, 1H), 7.35 (m, 2H), 6.66 (dd, J=7.8, 1.3 Hz, 1H), 6.55(dd, J=7.8, 6.6 Hz, 1H), 5.93 (s, 1H), 3.89 (s, 1H). ¹³C NMR (100 MHz,CDCl₃) δ 173.08, 158.99, 140.81, 140.20, 134.14, 131.72, 128.61, 128.42,127.35, 127.28, 126.83, 111.62, 109.67, 104.80, 58.52, 56.58. HRMS (EI)calcd for C₁₉H₁₇NOS [M]⁺ 307.1031. found 307.1029.

1-Benzyl-3-hydroxypyridine-2-thione (144b)

Reaction of 143b (0.050 g, 0.21 mmol) with 1M BBr₃ (0.66 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 142a afforded pure 144b(0.034 g, 72%) as olive green solid. ¹H NMR (400 MHz, cd₃od) δ 7.34 (m,3H), 6.84 (m, 1H), 5.83 (d, J=49.5 Hz, 1H). ¹³C NMR (100 MHz, CD₃OD) δ128.12, 127.61, 127.27, 126.94, 113.77. HRMS (EI) calcd for C₁₂H₁₁NOS[M]⁺ 217.0561. found 217.0762.

1-(1,1′-Biphenylmethyl)-3-hydroxypyridine-2-thione (144c)

Reaction of 143c (0.10 g, 0.32 mmol) with 1M BBr₃ (0.48 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 142a afforded pure 144c(0.074 g, 90%) as olive green solid. ¹H NMR (400 MHz, cd₃od) δ 7.39 (m,9H), 6.88 (m, 3H), 5.86 (s, 2H). ¹³C NMR (101 MHz, cd₃od) δ 141.33,140.21, 128.71, 128.52, 127.49, 127.45, 126.89. HRMS (EI) calcd forC₁₈H₁₅NOS [M]⁺ 293.0874. found 293.0873.

1-(4-Bromobenzyl)-3-methoxypyridine-2 (1H)-one (145)

To a stirring reaction mixture of 3-methoxypyridine-2-one (2.00 g, 16mmol) and K₂CO₃ (4.42 g, 32 mmol) in THF was added 4-bromobenzyl bromide(5.20 g, 20.8 mmol) slowly. Reaction mixture was the heated to refluxand stirring continued overnight. Reaction mixture was then cooled downand separated into organic and aqueous layer by adding CH₂Cl₂ (120 mL)and water (60 mL). Organic layer was separated and subsequently washedwith water (2×60 mL), brine (1×40 mL). Organic layer was dried on Na₂SO₄and solvent was evaporated in vacuo. Crude yellowish solid wastriturated with hexanes to give pure white solid 145 (3.49 g, 74%)without further purification. ¹H NMR (400 MHz, CDCl₃) δ 7.25 (m, 2H),7.04 (m, 2H), 6.78 (dd, J=6.8, 1.2 Hz, 1H), 6.45 (dd, J=7.2, 1.6 Hz,1H), 5.94 (t, J=7.2 Hz, 1H), 4.95 (s, 2H), 3.64 (s, 3H). ¹³C NMR (100MHz, CDCl₃) δ 157.71, 149.91, 135.29, 131.49, 129.59, 127.53, 121.57,111.82, 104.98, 55.54, 51.10. HRMS (EI) calcd for C₁₃H₁₂BrNO₂ [M]⁺293.0051. found 293.0051.

1-(4-Cyano-(1,1′-biphenylmethyl))-3-methoxyoxypyridine-2-one (146a)

145 (0.26 g, 0.86 mmol), (4-cyanophenyl)boronic acid (0.14 g, 0.95mmol), 2M aq. K₂CO₃ (0.24 g, 1.73 mmol), toluene (8 mL), EtOH (4 mL) andwater (4 mL) were added into reaction flask equipped with magneticstirrer and water condenser. The resulting suspension was degassed for10 min by sparging with argon gas. Pd(PPh₃)₄ (2.5 mol %) was added andthe reaction mixture was heated to reflux overnight under argonatmosphere. After cooling to room temperature, CH₂Cl₂ was added (50 mL)& washed with water (1×40 mL), brine (1×20 mL) and dried on Na₂SO₄.Column chromatography using gradient CHCl₃:Acetone:MeOH (with 5%-20% ofacetone and 1%-8% MeOH gradual increase) gave pure off white solid 146a(0.42 g, 78%). ¹H NMR (CDCl₃, 400 MHz) δ 7.60 (m, 4H), 7.40 (m, 4H),6.90 (dd, J=6.8, 1.6 Hz, 1H), 6.55 (dd, J=7.6, 1.6 Hz, 1H), 6.07 (t,J=7.2 Hz, 1H), 5.16 (s, 2H), 3.75 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ158.08, 150.28, 144.96, 138.62, 137.13, 132.56, 131.96, 128.85, 128.55,128.43, 127.96, 127.60, 127.53, 118.88, 112.11, 110.88, 105.24, 56.20,51.80. HRMS (EI) calcd for C₂OH₆N₂O₂ [M]⁺ 316.1212. found 316.1210.

1-(3-Cyano-(1,1′-biphenylmethyl))-3-methoxyoxypyridine-2-one (146b)

Reaction of 140 (0.25 g, 0.85 mmol), (3-cyanophenyl)boronic acid (0.14g, 0.93 mmol), 2M aq. K₂CO₃ (0.23 g, 1.69 mmol) and Pd(PPh₃)₄ (2.5 mol%) according to method described for synthesis of 146a within 18 hafforded 175 mg of 146b (66%) of white solid. ¹H NMR (400 MHz, CDCl₃) δ7.70 (m, 2H), 7.48 (m, 6H), 6.92 (dd, J=6.9, 1.6 Hz, 1H), 6.57 (dd,J=7.4, 1.4 Hz, 1H), 6.08 (t, J=7.2 Hz, 1H), 5.16 (s, 2H), 3.76 (s, 3H).¹³C NMR (100 MHz, CDCl₃) δ 158.09, 150.24, 141.70, 138.33, 136.81,131.38, 130.76, 130.45, 129.66, 128.87, 127.96, 127.36, 118.76, 112.82,112.14, 105.26, 55.84, 51.66. HRMS (EI) calcd for C₂₀H₁₆N₂O₂ [M]⁺316.1212. found 332.1216.

1-(2-Cyano-(1,1′-biphenylmethyl)-3-methoxyoxypyridine-2-one (146c)

Reaction of 140 (0.15 g, 0.50 mmol), (2-cyanophenyl)boronic acid (0.09g, 0.60 mmol), 2M aq. K₂CO₃ (0.14 g, 1.69 mmol) and Pd(PPh₃)₄ (2.5 mol%) according to method described for synthesis of 146a within 18 hafforded 118 mg of 146c (75%) of white solid. ¹H NMR (400 MHz, cdcl₃) δ7.72 (m, 1H), 7.60 (m, 1H), 7.45 (m, 2H), 6.94 (dd, J=6.9, 1.7 Hz, 1H),6.60 (dd, J=7.4, 1.6 Hz, 1H), 6.10 (t, J=7.2 Hz, 1H), 5.21 (s, 1H), 3.80(s, 1H). ¹³C NMR (100 MHz, cdcl₃) δ 158.01, 150.18, 144.70, 137.56,136.94, 133.61, 132.78, 129.91, 129.01, 128.31, 127.92, 127.55, 118.54,112.01, 110.94, 105.13, 55.74, 51.60. HRMS (EI) calcd for C₂₀H₁₆N₂O₂[M]⁺ 316.1212. found 316.1201.

1-(4-Methyl-(1,1′-biphenylmethyl))-3-methoxyoxypyridine-2-one (146d)

Reaction of 145 (0.25 g, 0.85 mmol), p-tolylboronic acid (0.14 g, 1.02mmol), 2M aq. K₂CO₃ (0.23 g, 1.69 mmol), Pd(PPh₃)₄ (2.5 mol %) accordingto method described for synthesis of 146a within 18 h afforded 259 mg of146d (quantitative) of white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.49 (d,J=8.1 Hz, 2H), 7.42 (d, J=8.1 Hz, 2H), 7.33 (d, J=8.1 Hz, 2H), 7.19 (d,J=8.0 Hz, 2H), 6.88 (dd, J=6.9, 1.6 Hz, 1H), 6.54 (dd, J=7.4, 1.4 Hz,1H), 6.03 (t, J=7.2 Hz, 1H), 5.15 (s, 2H), 3.76 (s, 3H), 2.34 (s, 3H).¹³C NMR (100 MHz, CDCl₃) δ 157.81, 149.88, 140.39, 137.29, 136.87,134.93, 129.21, 128.35, 127.66, 126.91, 126.53, 111.75, 104.78, 55.51,51.31, 20.80. HRMS (EI) calcd for C₂₀H₁₉NO₂ [M]⁺ 305.1416. found305.1422.

1-(3-Methyl-(1,1′-biphenylmethyl))-3-methoxyoxypyridine-2-one (146e)

Reaction of 140 (0.20 g, 0.68 mmol), m-tolylboronic acid (0.11 g, 0.82mmol), 2M aq. K₂CO₃ (0.19 g, 1.36 mmol) and Pd(PPh₃)₄ (2.5 mol %)according to method described for synthesis of 146a within 18 h afforded259 mg of 146e (quantitative) of white solid. ¹H NMR (400 MHz, cdcl₃) δ7.49 (m, 1H), 7.29 (m, 2H), 7.11 (d, J=7.2 Hz, 1H), 6.88 (dd, J=6.9, 1.7Hz, 1H), 6.53 (dd, J=7.4, 1.6 Hz, 1H), 6.03 (t, J=7.2 Hz, 1H), 5.15 (s,11H), 3.75 (s, 1H), 2.35 (s, 1H). ¹³C NMR (100 MHz, cdcl₃) δ 157.81,149.86, 140.58, 140.16, 137.99, 135.11, 128.37, 128.29, 127.83, 127.66,127.46, 127.10, 123.80, 111.81, 104.80, 55.48, 51.30, 21.19. HRMS (EI)calcd for C₂₀H₁₉NO₂ [M]⁺ 305.1416. found 305.1415.

1-(2-Methyl-(1,1′-biphenylmethyl))-3-methoxyoxypyridine-2-one (146f)

Reaction of 140 (0.20 g, 0.68 mmol), O-tolylboronic acid (0.11 g, 0.82mmol), 2M aq. K₂CO₃ (0.19 g, 1.36 mmol) and Pd(PPh₃)₄ (2.5 mol %)according to method described for synthesis of 146a within 18 h afforded243 mg of 146f (98%) of white solid. ¹H NMR (400 MHz, cdcl₃) δ 7.31 (d,J=7.9 Hz, 1H), 7.17 (m, 2H), 6.93 (m, 1H), 6.56 (dd, J=7.4, 1.6 Hz, 1H),6.06 (t, J=7.2 Hz, 1H), 5.18 (s, 1H), 3.76 (s, 1H), 2.20 (s, 1H). ¹³CNMR (101 MHz, cdcl₃) δ 158.13, 150.23, 141.51, 141.26, 135.23, 135.00,130.33, 129.68, 129.51, 128.13, 127.85, 127.34, 125.77, 112.13, 105.07,55.82, 51.79, 20.45. HRMS (EI) calcd for C₂₀H₁₉NO₂ [M]⁺ 305.1416. found305.1419.

1-(4-Dimethylamino-(1,1′-biphenylmethyl))-3-methoxyoxypyridine-2-one(146g)

Reaction of 145 (0.25 g, 0.85 mmol), (4-(dimethylamino)phenyl)boronicacid (0.17 g, 1.02 mmol), 2M aq. K₂CO₃ (0.23 g, 1.69 mmol), Pd(PPh₃)₄(2.5 mol %) according to method described for synthesis of 146a within18 h afforded 230 mg of 146 g (81%) as white solid. ¹H NMR (400 MHz,CDCl₃) δ 7.46 (m, 4H), 7.29 (m, 2H), 6.86 (dd, J=7.2, 2.0 Hz, 1H), 7.73(m, 2H), 6.52 (dd, J=7.2, 1.6 Hz, 1H), 6.01 (t, J=7.2 Hz, 1H), 5.13 (s,2H), 3.76 (s, 3H), 2.93 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 157.86,149.90, 149.73, 140.55, 133.81, 128.43, 128.04, 127.68, 127.28, 126.16,112.40, 111.77, 104.69, 55.53, 51.31, 40.18. HRMS (EI) calcd forC₂₁H₂₂N₂O₂ [M]⁺ 334.1681. found 334.1684.

1-(4-(6-(Dimethylamino)pyridine-3-yl)benzyl))-3-methoxyoxypyridine-2-one(146h)

Reaction of 145 (0.43 g, 1.44 mmol),(6-(dimethylamino)pyridine-3-yl)boronic acid (0.2 g, 1.20 mmol), 2M aq.K₂CO₃ (0.33 g, 2.41 mmol), Pd(PPh₃)₄ (2.5 mol %) according to methoddescribed for synthesis of 146a within 18 h afforded 335 mg of 146h(83%) of white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J=2.3 Hz, 1H),7.51 (dd, J=8.8, 2.5 Hz, 1H), 7.33 (d, J=8.2 Hz, 2H), 7.22 (d, J=8.2 Hz,2H), 6.80 (dd, J=6.9, 1.6 Hz, 1H), 6.44 (m, 2H), 5.94 (t, J=7.2 Hz, 1H),5.04 (s, 2H), 3.66 (s, 3H), 2.97 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ158.12, 157.61, 149.68, 145.49, 137.75, 135.14, 134.20, 128.35, 127.54,125.69, 123.15, 111.62, 105.23, 104.62, 55.36, 51.16, 37.66. HRMS (EI)calcd for C₂₀H₂₁N₃O₂ [M]⁺ 335.1634. found 335.1635.

1-(4-(Pyridin-4-yl)benzyl)-3-methoxy-pyridine-2-one (146i)

Reaction of 145 (0.25 g, 0.85 mmol), pyrdin-4-ylboronic acid (0.12 g,1.02 mmol), 2M aq. K₂CO₃ (0.23 g, 1.69 mmol), Pd(PPh₃)₄ (2.5 mol %)according to method described for synthesis of 146a within 18 h afforded203 mg of 146i (82%) of white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.54 (dd,J=4.5, 1.6 Hz, 2H), 7.48 (m, 2H), 7.36 (m, 4H), 6.87 (dd, J=6.9, 1.7 Hz,1H), 6.53 (dd, J=7.4, 1.6 Hz, 1H), 6.03 (t, J=7.2 Hz, 1H), 5.13 (s, 2H),3.72 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 157.82, 150.32, 150.01, 149.90,147.40, 137.31, 137.28, 128.57, 127.68, 127.04, 121.22, 111.90, 104.98,55.57, 51.42. HRMS (EI) calcd for C₁₈H₁₆N₂O₂ [M]⁺ 292.1212. found292.1205.

1-(4-Cyano-(1,1′-biphenylmethyl))-3-methoxyoxypyridine-2-thione (147a)

A suspension of 146a (0.13 g, 0.42 mmol) and Lawesson's reagent (0.10 g,0.25 mmol) in toluene (10 mL) was heated to reflux overnight. Reactionmixture was then cooled to room temperature. Toluene was evaporated offunder vacuo and resulting crude solid was directly loaded on prep-TLC.Elution with CHCl₃:Acetone:EtOH (12:1:0.2) gave 147a 123 mg (88%) ofyellow solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.60 (m, 4H), 7.40 (m, 4H), 6.90(dd, J=6.8, 1.6 Hz, 1H), 6.55 (dd, J=7.6, 1.6 Hz, 1H), 6.07 (t, J=7.2Hz, 1H), 5.16 (s, 2H), 3.75 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 158.08,150.28, 144.96, 138.62, 137.13, 132.56, 131.96, 128.85, 128.55, 128.43,127.96, 127.60, 127.53, 118.88, 112.11, 110.88, 105.24, 56.20, 51.80HRMS (EI) calcd for C₂₀H₁₆N₂OS [M]⁺ 332.0983. found 332.0987.

1-(3-Cyano-(1,1′-biphenylmethyl))-3-methoxypyridine-2-thione (147b)

Reaction of 146b (0.11 g, 0.36 mmol) and Lawesson's reagent (0.09 g,0.22 mmol) in toluene according to method described for synthesis of147a afforded 113 mg of 147b (95%) of yellow solid. ¹H NMR (400 MHz,DMSO-d₆) δ 7.92 (m, 3H), 7.84 (m, 2H), 7.71 (d, J=8.4 Hz, 2H), 7.36 (d,J=8.8 Hz, 2H), 7.00 (m, 1H), 6.80 (m, 1H), 5.95 (s, 2H), 3.78 (s, 3H).¹³C NMR (100 MHz, CDCl₃) δ 173.39, 159.26, 144.82, 138.81, 135.79,132.55, 131.81, 128.65, 127.58, 127.50, 118.77, 111.80, 110.98, 109.69,58.63, 56.74. HRMS (EI) calcd for C₂₀H₁₆N₂OS [M]⁺ 332.0983. found332.0984.

1-(2-Cyano-(1,1′-biphenylmethyl)-3-methoxyoxypyridine-2-thione (147c)

Reaction of 146c (0.09 g, 0.28 mmol) and Lawesson's reagent (0.07 g,0.17 mmol) in toluene according to method described for synthesis of147a afforded 72 mg of 147c (77%) of yellow solid. ¹H NMR (400 MHz,cdcl₃) δ 7.74 (m, 1H), 7.63 (td, J=7.7, 1.4 Hz, 1H), 7.45 (m, 2H), 6.71(dd, J=7.8, 1.3 Hz, 1H), 6.62 (dd, J=7.8, 6.6 Hz, 1H), 6.00 (s, 1H),5.28 (s, 1H), 3.93 (s, 1H). ¹³C NMR (101 MHz, cdcl₃) δ 173.38, 159.22,144.68, 137.79, 135.80, 133.67, 132.86, 131.95, 129.97, 129.17, 128.25,127.66, 118.59, 111.84, 111.01, 109.76, 58.63, 56.74. HRMS (EI) calcdfor C₂₀H₁₆N₂OS [M]⁺ 332.0983. found 332.0981.

1-(4-Methyl-(1,1′-biphenylmethyl))-3-methoxyoxypyridine-2-thione (147d)

Reaction of 146d (0.12 g, 0.39 mmol) and Lawesson's reagent (0.09 g,0.23 mmol) in toluene according to method described for synthesis of147a afforded 117 mg of 147d (94%) as yellow solid. ¹H NMR (400 MHz,CDCl₃) δ 7.51 (m, 2H), 7.42 (m, 2H), 7.34 (m, 3H), 7.21 (dd, J=8.4, 0.6Hz, 2H), 6.66 (dd, J=7.8, 1.2 Hz, 1H), 6.55 (dd, J=7.8, 6.6 Hz, 1H),5.92 (s, 2H), 3.89 (s, 3H), 2.36 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ173.05, 158.97, 140.74, 137.30, 137.08, 133.81, 131.70, 129.33, 128.42,127.12, 126,64, 111.59, 109.65, 58.52, 56.57, 20.91. HRMS (EI) calcd forC₂₀H₁₉NOS [M]⁺ 321.1187. found 321.1192.

1-(3-Methyl-(1,1′-biphenylmethyl))-3-methoxyoxypyridine-2-thione (147e)

Reaction of 146e (0.12 g, 0.37 mmol) and Lawesson's reagent (0.09 g,0.23 mmol) in toluene according to method described for synthesis of147a afforded 112 mg of 147e (93%) as yellow solid. ¹H NMR (400 MHz,cdcl₃) δ 7.53 (m, 1H), 7.33 (m, 2H), 7.14 (dd, J=7.1, 0.6 Hz, 1H), 6.67(dd, J=7.8, 1.2 Hz, 1H), 6.56 (m, 1H), 5.94 (s, 1H), 3.91 (s, 1H), 2.39(s, 1H). ¹³C NMR (101 MHz, cdcl₃) δ 173.21, 159.07, 141.06, 140.28,138.24, 134.07, 131.72, 128.57, 128.48, 128.09, 127.69, 127.44, 124.01,111.62, 109.66, 58.60, 56.63, 21.39. HRMS (EI) calcd for C₂₀H₁₉NOS [M]⁺305.1187. found 321.1188.

1-(2-Methyl-(1,1′-biphenylmethyl)-3-methoxyoxypyridine-2-thione (147f)

Reaction of 146f (0.14 g, 0.45 mmol) and Lawesson's reagent (0.11 g,0.27 mmol) in toluene according to method described for synthesis of147a afforded 118 mg of 147f (86%) as yellow solid. ¹H NMR (400 MHz,cdcl₃) δ 7.42 (dd, J=6.6, 1.0 Hz, 1H), 7.22 (m, 3H), 6.68 (d, J=7.3 Hz,1H), 6.59 (m, 1H), 5.96 (s, 1H), 3.90 (s, 1H), 2.22 (s, 1H). ¹³C NMR(101 MHz, cdcl₃) δ 158.97, 141.54, 140.93, 135.02, 133.59, 131.79,130.13, 129.46, 129.42, 127.65, 127.18, 125.57, 111.62, 109.67, 58.56,56.57, 20.25. HRMS (EI) calcd for C₂₀H₁₉NOS [M]⁺ 305.1187. found321.1189.

1-(4-Dimethylamino-(1,1′-biphenylmethyl))-3-methoxyoxypyridine-2-thione(147g)

Reaction of 146 g (0.22 g, 0.67 mmol) and Lawesson's reagent (0.16 g,0.40 mmol) in toluene according to method described for synthesis of147a afforded 172 mg of 147 g (73%) as yellow solid. ¹H NMR (400 MHz,CDCl₃) 7.39 (m, 5H), 7.22 (m, 2H), 6.69 (m, 3H), 6.65 (m, 1H), 5.82 (s,2H), 3.82 (s, 3H), 2.88 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 172.36,158.73, 149.92, 140.89, 132.48, 131.84, 128.44, 128.20, 127.29, 126.32,112.69, 112.02, 110.17, 58.68, 56.31, 40.24. HRMS (EI) calcd forC₂₁H₂₂N₂OS [M]⁺ 350.1453. found 350.1451.

1-(4-(6-(Dimethylamino)pyridin-3-yl)benzyl))-3-methoxyoxypyridine-2-thione(147h)

Reaction of 146h (0.14 g, 0.42 mmol) and Lawesson's reagent (0.10 g,0.25 mmol) in toluene according to method described for synthesis of147a afforded 130 mg of 147h (88%) of yellow solid. ¹H NMR (400 MHz,CDCl₃) δ 8.32 (d, J=2.3 Hz, 1H), 7.56 (dd, J=8.6, 2.1 Hz, 1H), 7.36 (dd,J=19.8, 7.3 Hz, 3H), 7.26 (d, J=8.0 Hz, 2H), 6.61 (d, J=7.8 Hz, 1H),6.50 (m, 2H), 5.85 (s, 2H), 3.83 (s, 3H), 3.03 (s, 6H). ¹³C NMR (100MHz, CDCl₃) δ 172.74, 158.80, 158.31, 145.65, 138.13, 135.32, 133.08,131.66, 128.46, 125.95, 123.21, 111.57, 109.64, 105.42, 58.42, 56.47,37.86. HRMS (EI) calcd for C₂₀H₂₁N₃OS [M]⁺ 351.1405. found 351.1405.

1-(4-(Pyridin-4-yl)benzyl)-3-methoxypyridine-2-thione (147i)

Reaction of 146i (0.13 g, 0.45 mmol) and Lawesson's reagent (0.11 g,0.27 mmol) in toluene according to method described for synthesis of147a afforded 104 mg of 147i (76%) as yellow solid with greenish tinge.¹H NMR (400 MHz, CDCl₃) δ 8.59 (d, J=5.9 Hz, 2H), 7.55 (d, J=8.3 Hz,2H), 7.39 (m, 5H), 6.67 (dd, J=7.8, 1.1 Hz, 1H), 6.58 (dd, J=73, 6.7 Hz,1H), 5.95 (s, 2H), 3.89 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 173.29,159.15, 150.11, 147.43, 137.67, 136.17, 131.78, 128.58, 127.28, 121.35,111.72, 109.67, 58.54, 56.66. HRMS (EI) calcd for C₁₈H₁₆N₂OS [M]⁺308.0983. found 308.0975.

1-(4-Cyano-(1,1′-biphenylmethyl)-3-hydroxyoxypyridine-2-one (148a)

To a solution of 146a (0.1 g, 0.32 mmol) in dry CH₂Cl₂ (8 mL) was slowlyadded 1M BBr₃ (0.35 mL) at −30° C. under argon atmosphere. The reactionmixture was stirred for 32 h at room temperature. The mixture was againcooled to −30 & then MeOH (5 mL) was slowly added to the mixture. Afterevaporation of solvent, the residue was adjusted to pH 7 with 1M NaOHand then extracted with CHCl₃ (3×30 mL). The combined organic layerdried over Na₂SO₄. After evaporation of solvent, the residue waspurified by prep-TLC with CHCl₃:Acetone:EtOH (10:1:0.2) to give 89 mg of148a (94%) as pure slightly brownish solid. ¹H NMR (400 MHz, DMSO) δ9.08 (s, 1H), 7.87 (dd, J=24.8, 7.9 Hz, 4H), 7.71 (d, J=7.6 Hz, 2H),7.40 (d, J=7.7 Hz, 2H), 7.29 (d, J=6.7 Hz, 1H), 6.70 (d, J=6.7 Hz, 1H),6.13 (t, J=6.8 Hz, 1H), 5.18 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 147.11,145.13, 139.24, 136.77, 132.87, 128.95, 127.97, 127.89, 127.06, 119.08,114.15, 111.35, 107.55, 52.48, 29.93. HRMS (ESI) calcd for C₁₉H₁₅N₂O₂[M+H]⁺ 303.1128. found 303.1124.

1-(3-Cyano-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-one (148b)

Reaction of 146b (0.05 g, 0.15 mmol) with 1M BBr₃ (0.26 mL) in dryCH₂Cl₂ within 48 h according to procedure 148a afforded pure 148b (44mg, quantitative) as slightly brownish solid. ¹H NMR (400 MHz, DMSO-d₆)δ 9.08 (s, 1H), 8.11 (s, 1H), 7.98 (d, J=7.8 Hz, 1H), 7.70 (m, 4H), 7.40(d, J=7.7 Hz, 2H), 7.28 (d, J=6.2 Hz, 1H), 6.70 (d, J=6.8 Hz, 1H), 6.13(t, J=6.7 Hz, 1H), 5.17 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 146.78,141.62, 138.62, 136.13, 131.31, 130.82, 130.51, 129.62, 128.71, 127.49,126.66, 118.66, 113.90, 112.96, 107.26, 52.25. HRMS (EI) calcd forC₁₉H₁₄N₂O₂ [M+H]⁺ 302.1055. found 302.1055.

1-(2-Cyano-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-one (148c)

Reaction of 146e (0.072 g, 0.15 mmol) with 1M BBr₃ (0.34 mL) in dryCH₂Cl₂ within 48 h according to procedure 148a afforded pure 148c (61mg, 90%) as slightly brownish solid. ¹H NMR (400 MHz, cdcl₃) δ 7.75 (d,J=7.6 Hz, 1H), 7.63 (t, J=7.7 Hz, 1H), 7.53 (d, J=7.3 Hz, 1H), 7.43 (m,2H), 7.12 (m, 1H), 6.86 (dd, J=28.2, 6.6 Hz, 1H), 6.19 (t, J=6.1 Hz,1H), 5.25 (s, 1H). ¹³C NMR (101 MHz, cdcl₃) δ 144.67, 137.88, 13642,133.71, 132.85, 129.95, 129.23, 128.20, 127.69, 118.56, 111.08, 107.17,52.13. HRMS (EI) calcd for C₁₉H₁₄N₂O₂ [M+H]⁺ 302.1055. found 302.1053.

1-(4-Methyl-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-one (148d)

Reaction of 146d (0.06 g, 0.20 mmol) with 1M BBr₃ (0.30 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 148a afforded pure 148d(57 mg, quantitative) as slightly brownish solid. ¹H NMR (400 MHz,DMSO-d₆) δ 9.07 (s, 1H), 7.57 (m, 2H), 7.50 (d, J=8.1 Hz, 2H), 7.34 (d,J=6.8 Hz, 2H), 7.25 (dd, J=14.8, 7.1 Hz, 3H), 6.69 (d, J=7.4 Hz, 1H),6.11 (t, J=7.1 Hz, 1H), 5.14 (s, 2H), 2.30 (s, 3H). ¹³C NMR (100 MHz,DMSO-d₆) δ 139.81, 137.29, 137.22, 136.68, 129.94, 128.77, 127.00,126.89, 51.69, 21.10. Even after repeated cycles, quaternary carbonscouldn't be visualized. (No improvement with relaxation delay of 2 sec).HRMS (EI) calcd for C₁₉H₁₇NO₂ [M]⁺ 291.1259. found 291.1250.

1-(3-Methyl-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-one (148e)

Reaction of 146e (0.064 g, 0.20 mmol) with 1M BBr₃ (0.30 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 148a afforded pure 148e(60 mg, quantitative) as slightly brownish solid. ¹H NMR (400 MHz,cdcl₃) δ 7.56 (d, J=7.5 Hz, 1H), 7.33 (m, 2H), 7.17 (d, J=7.2 Hz, 1H),6.85 (dd, J=22.2, 6.4 Hz, 1H), 6.16 (t, J=6.5 Hz, 1H), 5.23 (s, 1H),2.42 (s, 1H). ¹³C NMR (101 MHz, cdcl₃) δ 146.71, 141.19, 140.38, 138.32,134.70, 128.63, 128.41, 128.16, 127.79, 127.56, 126.60, 124.11, 113.66,106.98, 52.10, 21.45. HRMS (EI) calcd for C₁₉H₁₇NO₂ [M]⁺ 291.1259. found291.1263.

1-(2-Methyl-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-one (148f)

Reaction of 146f (0.077 g, 0.25 mmol) with 1M BBr₃ (0.38 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 148a afforded pure 148f(65 mg, 90%) as slightly brownish solid. ¹H NMR (400 MHz, cdcl₃) δ 7.28(m, 3H), 6.87 (dd, J=31.4, 6.8 Hz, 1H), 6.18 (t, J=6.9 Hz, 1H), 5.27 (d,J=16.7 Hz, 1H), 2.26 (s, 1H). ¹³C NMR (101 MHz, cdcl₃) δ 146.73, 141.81,141.14, 135.24, 134.30, 130.31, 129.66, 127.68, 127.37, 126.73, 125.75,113.59, 106.99, 52.21, 20.40. HRMS (EI) calcd for C₁₉H₁₇NO₂ [M]⁺291.1259. found 291.1260.

1-(4-Dimethylamino-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-one(148g)

Reaction of 146g (0.10 g, 0.30 mmol) with 1M BBr₃ (0.45 mL) in dryCH₂Cl₂ (8 mL) within 48 h according to procedure 148a afforded pure 148g(84 mg, 87%) as slightly brownish solid. ¹H NMR (400 MHz, DMSO-d₆) δ9.06 (s, 1H), 7.52 (d, J=8.0 Hz, 3H), 7.28 (m, 4H), 6.76 (m, 2H), 6.10(t, J=5.8 Hz, 1H), 5.12 (s, 2H), 2.90 (s, 6H). ¹³C NMR (100 MHz, CDCl₃)δ 158.71, 149.98, 146.62, 141.07, 135.81, 133.29, 128.83, 128.47,128.21, 128.07, 127.98, 127.55, 126.57, 113.48, 112.60, 106.89, 52.11,40.44. HRMS (EI) calcd for C₂₀H₂₀N₂O₂ [M]⁺ 320.1524. found 320.1512.

1-(4-(6-(Dimethylamino)pyridine-3-yl)benzyl))-3-hydroxyoxypyridine-2-one(148h)

Reaction of 146h (0.10 g, 0.29 mmol) with 1M BBr₃ (0.45 mL) in dryCH₂Cl₂ (7 mL) within 48 h according to procedure 148a afforded pure 148h(67 mg, 83%) as slightly brownish solid. ¹H NMR (400 MHz, CDCl₃) δ 8.40(s, 1H), 7.66 (d, J=8.0 Hz, 1H), 7.49 (d, J=7.7 Hz, 2H), 7.33 (d, J=7.7Hz, 2H), 6.84 (dd, J=27.3, 6.8 Hz, 3H), 6.57 (d, J=8.7 Hz, 1H), 6.15 (t,J=7.0 Hz, 1H), 5.20 (s, 2H), 3.12 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ158.47, 146.67, 145.66, 138.31, 135.79, 133.89, 131.95, 128.49, 126.79,126.28, 123.61, 114.16, 107.19, 105.94, 52.13, 38.15. HRMS (EI) calcdfor C₁₉H₁₉N₃O₂ [M]⁺ 321.1477. found 321.1479.

1-(4-(Pyridine-4-yl)benzyl)-3-hydroxypyridine-2-one (148i)

Reaction of 146i (0.10 g, 0.34 mmol) with 1M BBr₃ (0.51 mL) in dryCH₂Cl₂ within 48 h according to procedure 148a afforded pure 148i (79mg, 83%) as slightly brownish solid. ¹H NMR (400 MHz, CDCl₃) δ 8.65 (s,2H), 7.46 (s, 2H), 7.39 (d, J=7.9 Hz, 2H), 6.83 (dd, J=22.5, 7.0 Hz,2H), 6.16 (t, J=7.0 Hz, 1H), 5.22 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ150.23, 147.62, 146.76, 137.98, 136.87, 128.66, 127.51, 126.64, 121.58,113.66, 107.13, 52.19. HRMS (FAB) calcd for C₁₇H₁₅N₂O₂ [M+H]⁺ 279.1133.found 279.1147.

1-(4-Cyano-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-thione (149a)

Reaction of 147a (0.10 g, 0.30 mmol) with 1M BBr₃ (0.33 mL) in dryCH₂Cl₂ (8 mL) within 48 h according to procedure 148a afforded pure 149a(84 mg, 88%) as olive green color solid. ¹H NMR (400 MHz, DMSO-d₆) δ7.87 (m, 5H), 7.74 (d, J=8.1 Hz, 2H), 7.37 (d, J=8.1 Hz, 2H), 7.04 (m,1H), 6.90 (d, J=8.1 Hz, 1H), 5.87 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ164.47, 144.66, 139.24, 134.64, 132.60, 128.56, 127.74, 127.62, 118.75,111.15, 61.79, 29.64. HRMS (EI) calcd for C₁₉H₁₅N₂OS [M]⁺ 318.0827.found 318.0828.

1-(3-Cyano-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-thione (149b)

Reaction of 147b (0.07 g, 0.21 mmol) with 1M BBr₃ (0.32 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 148a afforded pure 149b(53 mg, 79%) as olive green color solid. ¹H NMR (400 MHz, CD₃OD) δ 7.89(m, 2H), 7.65 (m, 5H), 7.44 (d, J=8.2 Hz, 2H), 7.02 (d, J=7.7 Hz, 2H),6.77 (m, 1H), 5.90 (s, 2H). ¹³C NMR (100 MHz, CD₃OD) δ 142.99, 140.04,132.82, 132.18, 131.69, 131.12, 130.03, 129.97, 129.89, 128.69, 128.59,119.83, 113.94, 106.40, 54.78. HRMS (EI) calcd for C₁₉H₁₄N₂O₂ [M+H]⁺318.0827. found 318.0827

1-(2-Cyano-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-thione (149c)

Reaction of 147c (0.042 g, 0.21 mmol) with 1M BBr₃ (0.19 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 148a afforded pure 149c(31 mg, 78%) as olive green color solid. ¹H NMR (400 MHz, cd₃od) δ 7.77(dd, J=7.7, 0.9 Hz, 1H), 7.67 (m, 2H), 7.48 (m, 4H), 7.01 (d, J=13.1 Hz,1H), 6.70 (m, 11.1), 5.90 (s, 1H). ¹³C NMR (101 MHz, cd₃od) δ 145.34,138.90, 134.40, 133.88, 130.75, 129.88, 128.90, 128.62, 119.17, 115.12,111.38. HRMS (EI) calcd for C₁₉H₁₄N₂O₂ [M+H]⁺ 318.0821. found 318.0827

1-(4-Methyl-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-thione (149d)

Reaction of 147d (0.06 g, 0.20 mmol) with 1M BBr₃ (0.30 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 148a afforded pure 149d(45 mg, 73%) as olive green color solid. ¹H —NMR (400 MHz, CD₃OD) δ 7.70(d, J=1.1 Hz, 1H), 7.63 (d, J=6.3 Hz, 1H), 7.55 (d, J=8.2 Hz, 2H), 7.40(m, 3H), 7.21 (d, J=8.1 Hz, 2H), 7.03 (dd, J=14.9, 7.8 Hz, 2H), 6.75 (t,J=7.0 Hz, 1H), 5.87 (s, 2H), 2.35 (s, 3H). Even after repeated cycles,quaternary carbons couldn't be visualized. (No improvement withrelaxation delay of 2 see). HRMS (EI) calcd for C₁₉H₁₇NOS [M]⁺ 307.1031.found 307.1022.

1-(3-Methyl-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-thione (149e)

Reaction of 147e (0.08 g, 0.20 mmol) with 1M BBr₃ (0.36 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 148a afforded pure 149e(51 mg, 70%) as olive green color solid. ¹H NMR (400 MHz, cd₃od) δ 7.53(m, 1H), 7.30 (m, 2H), 7.12 (d, J=7.2 Hz, 1H), 6.97 (m, 1H), 6.68 (m,1H), 5.81 (s, 1H), 2.36 (s, 1H). ¹³C NMR (101 MHz, cd₃od) δ 141.53,140.21, 138.34, 128.62, 128.48, 128.21, 127.69, 127.54, 124.03, 21.19.HRMS (EI) calcd for C₁₉H₁₇NOS [M]⁺ 307.1031. found 307.1031.

1-(2-Methyl-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-thione (149f)

Reaction of 147f (0.08 g, 0.20 mmol) with 1M BBr₃ (0.36 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 148a afforded pure 149f(49 mg, 67%) as olive green color solid. ¹H NMR (400 MHz, cd₃od) δ 7.54(m, 1H), 7.19 (m, 3H), 6.98 (m, 1H), 6.70 (m, 1H), 5.83 (s, 1H), 2.19(s, 1H). ¹³C NMR (101 MHz, cd₃od) δ 142.14, 140.99, 135.05, 130.21,129.58, 129.48, 127.76, 127.33, 125.66, 20.02. HRMS (EI) calcd forC₁₉H₁₇NOS [M]⁺ 307.1031. found 307.1034.

1-(4-Dimethylamino-(1,1′-biphenylmethyl))-3-hydroxyoxypyridine-2-thione(149g)

Reaction of 147g (0.11 g, 0.33 mmol) with 1M BBr₃ (0.39 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 148a afforded pure 149g(68 mg, 62%) as olive green color solid. ¹H NMR (400 MHz, CDCl₃) δ 8.57(d, J=8.5 Hz, 1H), 7.53 (d, J=8.2 Hz, 2H), 7.46 (m, 2H), 7.33 (d, J=8.0Hz, 3H), 6.97 (d, J=6.8 Hz, 1H), 6.77 (d, J=8.8 Hz, 2H), 6.62 (m, 1H),5.79 (s, 2H), 2.98 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 169.71, 155.14,150.08, 141.51, 131.92, 130.80, 129.00, 128.83, 127.99, 127.58, 126.68,113.58, 112.62, 111.90, 60.11, 40.45. HRMS (EI) calcd for C₂₀H₂₀N₂OS[M]⁺ 336.1296. found 336.1295.

1-(4-(6-(Dimethylamino)pyridin-3-yl)benzyl))-3-hydroxyoxypyridine-2-thione(149h)

Reaction of 147h (0.064 g, 0.18 mmol) with 1M BBr₃ (0.27 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 148a afforded pure 149h(48 mg, 79%) as olive green color solid. ¹H NMR (400 MHz, CDCl₃) δ 8.30(s, 1H), 7.70 (d, J=8.9 Hz, 1H), 7.46 (s, 2H), 7.31 (t, J=7.6 Hz, 3H),6.98 (d, J=7.8 Hz, 1H), 6.67 (m, 2H), 5.75 (s, 2H), 3.11 (s, 8H). ¹³CNMR (100 MHz, CDCl₃) δ 157.02, 143.45, 142.18, 137.74, 136.98, 132.81,128.75, 127.56, 126.37, 123.70, 114.43, 107.03, 38.48, 29.54. HRMS (EI)calcd for C₁₉H₁₉N₃OS [M]⁺ 337.1249. found 337.1251.

1-(4-(Pyridin-4-yl)benzyl)-3-hydroxypyridine-2-thione (149i)

Reaction of 147i (0.08 g, 0.25 mmol) with 1M BBr₃ (0.38 mL) in dryCH₂Cl₂ (5 mL) within 48 h according to procedure 148a afforded pure 149i(65 mg, 88%) as olive green color solid. ¹H NMR (400 MHz, CDCl₃) δ 8.65(d, J=5.3 Hz, 2H), 8.55 (s, 1H), 7.62 (d, J=8.0 Hz, 2H), 7.42 (m, 5H),6.99 (d, J=7.5 Hz, 1H), 6.67 (t, J=7.0 Hz, 1H), 5.85 (s, 2H). ¹³C NMR(100 MHz, CDCl₃) δ 170.05, 155.32, 150.25, 147.44, 138.22, 135.48,130.91, 128.80, 127.55, 121.48, 113.73, 111.94, 59.91. HRMS (EI) calcdfor C₁₇H₁₄N₂OS [M]⁺ 294.0827. found 294.0823.

1-Propargyl-3-methoxypyridine-2-one (150)

To a stirring solution of 3-methoxy-2-pyridinone (1.00 g, 8.00 mmol) inDMF (25 mL), was added propargyl bromide (80 wt % solution in toluene,1.5 equiv) and K₂CO₃ (3.313 g, 24 mmol). Reaction mixture was heated to100° C. After overnight reaction, reaction mixture was partitionedbetween EtOAc (120 mL) and water (100 mL). Organic layer was separatedand washed repeatedly with water (7×100 mL) and brine (1×60 mL). Organiclayer was dried on Na₂SO₄ and evaporated under vacuo to yield dark browncrude product. Column chromatography using gradient CH₂Cl₂:Acetone (max20%) gave pure 150 (0.79 g, 60%) as a slightly brownish solid. ¹H NMR(CDCl₃, 400 MHz) δ 2.36 (1H, t, J=2.8), 3.67 (3H, m), 4.66 (1H, d,J=2.8), 6.05 (1H, t, J=7.2), 6.50 (1H, dd, J=1.6, 7.2), 7.11 (1H, dd,J=1.6, 6.8); ¹³C NMR (CDCl₃, 100 MHz) δ 37.2, 55.5, 74.6, 77.3, 104.8,112.0, 126.3, 149.4, 157.1. HRMS (EI) calcd for C₉H₉NO₂ [M]⁺ 163.0633.found 163.0636

1-Phenyltriazolylmethyl-3-methoxypyridine-2-one (151a)

150 (0.32 g, 1.951 mmol) and phenylazide (0.348 g, 2.926 mmol) weredissolved in anhydrous THF (10 mL) and stirred under argon at roomtemperature. Copper (I) iodide (0.011 g, 0.07 mmol) and Hunig's base(0.1 mL) were then added to the reaction mixture, and stirring continuedfor 4 h. The reaction mixture was diluted with CH₂Cl₂ (40 mL) and washedwith 1:4 NH₄OH/saturated NH₄Cl (3×30 mL) and saturated NH₄Cl (30 mL).The organic layer was dried over Na₂SO₄ and concentrated in vacuo. Thecrude product was triturated with hexanes to give 510 mg (92%) of whitesolid 151a. ¹H NMR (CDCl₃, 400 MHz) δ 3.58 (3H, s), 5.11 (2H, s), 5.93(1H, t, J=7.6), 6.42 (1H, d, J=7.2), 7.07 (1H, d, J=6.4), 7.16-7.20 (1H,m), 7.26 (2H, t, J=7.6), 7.49 (2H, d, J=8.0); ¹³C NMR (CDCl₃, 100 MHz) δ44.2, 55.2, 104.7, 112.0, 119.7, 121.8, 127.9, 128.1, 129.0, 136.2,142.8, 149.3, 157.2. HRMS (EI) calcd for C₁₅H₁₄N₄O₂ [M]⁺ 282.1117. found282.1115.

1-(4-Dimethylamino)phenylltriazolylmethyl-3-methoxypyridine-2-one (151b)

Reaction of 4-azido-N,N-dimethylaniline (0.09 g, 0.61 mmol) and 150(0.10 g, 0.61 mmol) within 4 h as described for synthesis of 151a gavecompound 151b (0.13 g, 68%) as a white solid. ¹H NMR (400 MHz, cdcl₃) δ8.04 (s, 1H), 7.41 (m, 2H), 7.15 (dd, J=6.9, 1.7 Hz, 1H), 6.62 (m, 2H),6.51 (dd, J=7.5, 1.6 Hz, 1H), 6.03 (m, 1H), 5.19 (s, 2H), 3.70 (s, 3H),2.89 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 157.54, 150.18, 149.65, 142.60,128.17, 126.18, 121.90, 121.43, 112.20, 111.84, 104.93, 55.55, 44.54,40.08. HRMS (EI) calcd for C₁₇H₁₉N₅O₂ [M]⁺ 325.1539. found 325.1544.

1-Phenyltriazolylmethyl-3-hydroxypyridine-2-one (152a)

Reaction of 151a (0.212 g, 0.75 mmol) and 1M BBr₃ in CH₂Cl₂ (1.5 equiv)within 48 h as described for the synthesis of 148a gave compound 152a(0.123 g, 61%) as light brown solid. ¹H NMR (DMSO-d₆, 400 MHz) δ 5.27(2H, s), 6.13 (1H, t, J=6.4), 6.67 (1H, d, J=7.2), 7.30 (1H, d, J=5.6),7.45-7.49 (1H, m), 7.57 (2H, t, J=7.6), 7.87 (2H, d, J=8.0), 8.73 (1H,s), 9.08 (1H, s); ¹³C NMR (101 MHz, CDCl₃) δ 136.59, 129.60, 128.94,122.29, 120.62, 107.99, 29.29. (quaternary carbons were not seen). HRMS(EI) calcd for C₁₄H₁₂N₄O₂ [M]⁺ 268.0960. found 268.0967.

1-(4-Dimethylamino)phenylltriazolylmethyl-3-hydroxypyridine-2-one (152b)

Reaction of 151b (0.045 g, 0.14 mmol) and 1M BBr₃ in CH₂Cl₂ (0.22 mL,1.5 equiv) within 48 h as described for the synthesis of 148a gavecompound 152b (0.033 g, 77%) as light brown solid. ¹H NMR (400 MHz,cdcl₃) δ 8.08 (s, 1H), 7.48 (dd, J=19.9, 8.4 Hz, 2H), 7.18 (d, J=6.7 Hz,1H), 6.69 (ddd, J=25.8, 24.8, 7.5 Hz, 4H), 6.16 (t, J=6.9 Hz, 1H), 5.31(s, 2H), 2.98 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 158.35, 150.52,146.68, 142.48, 127.10, 126.44, 122.06, 121.88, 121.76, 112.12, 107.14,44.66, 40.33. HRMS (EI) calcd for C₁₆H₁₇N₅O₂ [M]⁺ 311.1382. found311.1386.

1-Phenyltriazolylmethyl-3-methoxypyridine-2-thione (153a)

Reaction of 151a (0.295 g, 1.04 mmol) and Lawesson's reagent (0.252 g,0.624 mmol) in toluene (15 mL) within 12 h as described for thesynthesis of 147a gave compound 153a (0.291 g, 94%) as yellow solid. ¹HNMR (CDCl₃, 400 MHz) δ 3.90 (3H, s), 6.04 (2H, s), 6.62-6.68 (2H, m),7.38-7.42 (1H, m), 7.48 (2H, t, J=7.2), 7.68 (2H, d, J=8.0), 7.82 (1H,d, J=4.8), 8.53 (1H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 50.9, 56.4, 110.0,112.0, 120.2, 122.4, 128.5, 129.3, 132.2, 136.4, 141.9, 158.7, 171.5HRMS (EI) calcd for C₁₅H₁₄N₄O₂ [M]⁺ 282.1117. found 282.1115. HRMS (EI)calcd for C₁₅H₁₄N₄OS [M]⁺ 298.0888. found 298.0888.

1-(4-Dimethylamino)phenylltriazolylmethyl-3-methoxypyridine-2-thione(153b)

Reaction of 151b (0.078 g, 0.248 mmol) and Lawesson's reagent (0.06 g,0.15 mmol) in toluene (8 mL) within 12 h as described for the synthesisof 147a gave compound 153b (0.07 g, 84%) as yellow solid. ¹H NMR (400MHz, cdcl₃) δ 8.36 (s, 1H), 7.81 (dd, J=6.4, 1.5 Hz, 1H), 7.46 (m, 2H),6.66 (m, 4H), 6.01 (s, 2H), 3.87 (s, 3H), 2.97 (s, 6H). ¹³C NMR (100MHz, CDCl₃) δ 171.82, 158.93, 150.44, 141.54, 132.44, 126.29, 122.49,121.77, 112.10, 112.03, 110.14, 56.65, 51.20, 40.29. HRMS (EI) calcd forC₁₇H₁₉N₅OS [M]⁺ 314.1310. found 314.1304.

1-Phenyltriazolylmethyl-3-hydroxypyridine-2-thione (154a)

Reaction of 153a (0.28 g, 0.93 mmol) and 1M BBr₃ in CH₂Cl₂ (1.5 equiv)within 48 h as described for the synthesis of 148a gave compound 154a(0.178 g, 67%) as olive green solid. ¹H NMR (400 MHz, CDCl₃) δ 8.43 (d,J=16.6 Hz, 1H), 8.39 (s, 1H), 7.79 (dd, J=6.6, 1.3 Hz, 1H), 7.69 (m,2H), 7.46 (m, 3H), 6.97 (dd, J=7.7, 1.2 Hz, 1H), 6.69 (dd, J=7.6, 6.8Hz, 1H), 5.94 (s, 2H). ¹³C NMR (100 MHz, CDC₃) δ 168.59, 155.12, 141.60,136.66, 131.65, 129.69, 128.99, 122.57, 120.60, 114.04, 112.47, 77.30,76.99, 76.67, 52.08. HRMS (EI) calcd for C₁₄H₁₂N₄OS [M]⁺ 284.0730. found284.0732.

1-(4-Dimethylamino)phenylltriazolylmethyl-3-hydroxypyridine-2-thione(154b)

Reaction of 153b (0.07 g, 0.212 mmol) and 1M BBr₃ in CH₂Cl₂ (0.64 mL,1.5 equiv) within 48 h as described for the synthesis of 148a gavecompound 154b (0.032 g, 48%) as olive green solid. ¹H NMR (400 MHz,CDCl₃) δ 8.42 (s, 1H), 8.29 (d, J=11.5 Hz, 1H), 7.79 (d, J=6.4 Hz, 1H),7.50 (m, 2H), 6.98 (dd, J=36.5, 28.9 Hz, 2H), 6.72 (m, 3H), 5.94 (s,2H), 3.01 (d, J=2.7 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 155.09, 150.66,141.13, 131.72, 127.68, 126.27, 122.45, 121.97, 114.04, 112.52, 112.16,52.21, 40.41. HRMS (EI) calcd for C₁₆H₁₇N₅OS [M]⁺ 327.1154. found327.1154.

Example 9 2^(nd) Generation 3-Hydroxypyridine-2-thione-based HDACInhibitors

Compounds with variable methylene group linker lengths; from one (asseen in compound 154a) to seven (as seen in SAHA) were synthesized andevaluated for their activity as HDAC inhibitors.

The synthesis of 2^(nd) generation 3-HPT-based compounds is depicted inScheme 20. The crucial azidoalkyl methanesulfonates are synthesized intwo steps. The reaction of corresponding bromoalkanols with sodium azidefurnished azidoalkanols 155a-155f which were subsequently mesylated togive methanesulfonate intermediates 156a-156f in almost quantitativeyields. As described earlier, N-alkylation of O-methyl or O-benzylprotected 3-hydroxypyridine-2-one with the mesylate intermediates gaveazido intermediates 157a-157f in moderate to good yields. Aromaticsurface recognition cap group was linked to these azido intermediatesvia 1,2,3-triazole ring. Cu(I)-catalyzed Huisgen cycloaddition betweenphenylacetylene and azido intermediates 157a-157f, followed bydeprotection of methyl ether by Lewis acid BBr₃ furnished the desiredcompounds in good to excellent yields. All the higher linker thionecompounds were synthesized from their carbonyl counterparts by usingP₄S₁₀ chemistry as previously reported. However, methylene linkercompound 159a did not tolerate the rigorous temperature conditions andwas degraded to complex reaction mixture within 2 hrs. Therefore thesynthetic scheme shown in Scheme 20 was modified. Incorporation ofO-methyl protected intermediate 158a permitted the use of milderLawesson's reaction chemistry to access thione compound 161 (Scheme 20).

Activities of 2^(nd) generation compounds also were consistent for HDAC6and HDAC8 selectivity over HDAC1 and thione compounds alwaysoutperformed their corresponding carbonyl compounds. It is interestingto note that very similar trend was observed for HDAC6 and HDAC8activities according to variation in linker length. For thione analogs,increase in methylene spacer by 1-carbon to compound 154a showssignificant loss of activity (comparing 154a and 161 in Table 11 andTable 12). Gradual increase in methylene spacer length from 3-carbon to5-carbon shows improvement in activity with 5-carbon linker showsoptimum activity (compound 162d, IC₅₀=911 nM for HDAC6; IC₅₀=917 nM forHDA8). However, one additional carbon linker again results in drop ofactivity (Table 12; comparing 162d and 162e). Interestingly, 7-carbonlinker compound 1621 showed superior activity than 162e however it stilldidn't surpass the activity of 162d.

This suggests that 5-carbon methylene chain is optimum linker in HDACidesign containing 3-HPT as ZBG. In addition to five methylene spacer,one and seven methylene spacer showed comparable activities and shouldbe viable leads as well.

TABLE 12 In vitro HDAC Inhibition of 2 _(nd) Generation Compounds IC₅₀(nM) COMPOUND HDAC1 HDAC6 HDAC8

  159a 12% NI 15%

  161 21% 14% 56%

  159b  8% 41% 47%

  162b 27% 3628 ± 1363 63%

  159c 15% NI 10%

  162c ND 1085 ± 333  3303 ± 260 

  159d 43% NI 30%

  162d 30% 911 ± 173 917 +/− 139

  159c 12% NI 5%

  162e 23% 22% 6791 ± 910 

  159f  2% 34% 42%

  162f 35% 955 ± 150 232 ± 19  SAHA 38 +/− 2 95% 232 ± 19  SAHA was usedfor a positive control; NI — No significant Inhibition (below 20%Inhibition); % inhibitions of the compounds at 10 μM are given if theIC₅₀ was above 10 μM.

Synthetic Procedures 2-Azidoethyl methanesulfonate (156a)

To a solution of compound 2-azidoethanol (1.00 g, 11.49 mmol) in THF (25mL) and triethylamine (Et₃N) (2.418 mL, 17.24 mmol) was added mesylchloride (1.328 mL, 17.24 mmol) at 0° C., and the mixture was allowed towarm to room temperature. Stirring continued for 3 h, during which TLCrevealed a quantitative conversion into a higher R_(f) product. CH₂Cl₂(70 mL) and saturated sodium bicarbonate (50 mL) were added, and the twolayers were separated. The organic layer was washed with sodiumbicarbonate (2×50 mL), saturated brine (45 mL) and dried over Na₂SO₄.Solvent was evaporated off to give crude compound 156a (1.70 g) as acolorless oil, which was used for next step without furtherpurification.

2-Azidopropyl methanesulfonate (156b)

Reaction of 3-azidopropanol (1.00 g, 9.90 mmol) and mesyl chloride (1.14mL, 14.85 mmol) within 3 h as described for synthesis of 156a gavecompound 156b (1.18 g) as a crude colorless oil.

2-Azidobutyl methanesulfonate (156c)

Reaction of 4-azidobutanol (0.80 g, 6.95 mmol) and mesyl chloride (1.48mL, 10.43 mmol) within 3 h as described for synthesis of 156a gavecompound 156c (1.33 g) as a crude colorless oil.

2-Azidopentyl methanesulfonate (156d)

Reaction of 5-azidopentanol (1.1 g, 8.53 mmol) and mesyl chloride (0.98mL, 12.79 mmol) within 3 h as described for synthesis of 156a gavecompound 156d (1.54 g) as a crude colorless oil.

2-Azidohexyl methanesulfonate (156e)

Reaction of 6-azidohexanol (0.74 g, 5.22 mmol) and mesyl chloride (0.61mL, 7.83 mmol) within 3 h as described for synthesis of 156a gavecompound 156e (0.87 g) as a crude colorless oil.

2-Azidoheptyl methanesulfonate (156f)

Reaction of 7-azidoheptanol (0.55 g, 3.49 mmol) and mesyl chloride (0.41mL, 5.30 mmol) within 3 h as described for synthesis of 156a gavecompound 1561 (0.79 g) as a crude colorless oil.

1-(2-Azidoethyl)-3-methoxypyridine-2-one (157a)

3-methoxypyridine-2-one (0.40 g, 3.20 mmol), 156a (0.79 g, 4.80 mmol),and K₂CO₃ (1.32 g, 9.60 mmol) stirred overnight in THF at refluxtemperature. Reaction mixture was cooled down to room temperature.Reaction mixture was partitioned between CH₂Cl₂ (60 mL) and water (50mL). Organic layer washed with water (2×35 mL), brine (1×30 mL). Organiclayer dried on Na₂SO₄ and evaporated in vacuo. Crude product waspurified by flash chromatography with stepwise gradient of CH₂Cl₂ andacetone (max 15%) to give 0.34 mg of 157a (55%) as colorless oil. ¹H NMR(400 MHz, CDCl₃) δ 6.77 (dd, J=6.9, 1.6 Hz, 1H), 6.48 (dd, J=7.5, 1.6Hz, 1H), 5.96 (m, 1H), 3.91 (m, 2H), 3.62 (s, 3H), 3.52 (m, 2H). ¹³C NMR(100 MHz, CDCl₃) δ 157.39, 149.41, 128.61, 112.16, 104.37, 55.36, 49.00,48.91. HRMS (EI) calcd for C₈H₁₀N4O₂ [M]⁺ 194.0804. found 194.0816.

(3-Azidopropyl)-3-benzyloxypyridine-2-one (157b)

Reaction of 3-benzyloxypyridine-2-one (0.40 g, 1.99 mmol) and 156b (0.53g, 2.98 mmol) within 16 h as described for synthesis of 157a gavecompound 157b (0.30 g, 53%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃)7.28 (m, 5H), 6.84 (dd, J=6.9, 1.6 Hz, 1H), 6.60 (dd, J=7.4, 1.5 Hz,1H), 5.98 (t, J=7.1 Hz, 1H), 3.97 (t, J=6.8 Hz, 2H), 3.28 (t, J=6.5 Hz,2H), 1.98 (p, J=6.7 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 157.78, 148.65,135.93, 128.85, 128.24, 127.69, 127.03, 115.16, 104.53, 70.39, 48.14,47.06, 27.66. HRMS (EI) calcd for C₁₅H₁₆N₄O₂ [M]⁺ 284.1273. found284.1271.

1-(4-Azidobutyl)-3-benzyloxypyridine-2-one (157c)

Reaction of 3-benzyloxypyridine-2-one (0.40 g, 1.99 mmol) and 156c (0.58g, 2.98 mmol) within 16 h as described for synthesis of 157a gavecompound 157c (0.36 g, 62%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃)δ 7.22 (m, 5H), 6.78 (dd, J=6.9, 1.7 Hz, 1H), 6.54 (dd, J=7.4, 1.7 Hz,1H), 5.91 (m, 1H), 4.96 (s, 2H), 3.85 (t, J=7.2 Hz, 2H), 3.17 (t, J=6.8Hz, 2H), 1.71 (m, 2H), 1.48 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 157.52,148.27, 135.78, 128.37, 127.98, 127.42, 126.82, 114.92, 104.32, 70.09,50.43, 48.46, 25.83, 25.42. HRMS (EI) calcd for C₁₆H₁₈N₄O₂ [M]⁺298.1430. found 298.1446.

1-(5-Azidopentyl)-3-benzyloxypyridine-2-one (157d)

Reaction of 3-benzyloxypyridine-2-one (0.50 g, 2.49 mmol) and 156d (0.62g, 2.98 mmol) within 16 h as described for synthesis of 157a gavecompound 157d (0.49 g, 63%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃)δ 7.35 (d, J=8.0 Hz, 2H), 7.23 (m, 3H), 6.81 (m, 1H), 6.56 (m, 1H), 5.94(m, 1H), 5.01 (s, 2H), 3.85 (m, 2H), 3.18 (m, 2H), 1.71 (m, 2H), 1.54(m, 2H), 1.32 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 157.68, 148.47,135.98, 128.60, 128.12, 127.53, 126.93, 115.09, 104.32, 70.27, 50.77,49.21, 28.19, 28.06, 23.37. HRMS (EI) calcd for C₁₇H₂₀N₄O₂ [M]⁺312.1586. found 312.1589.

1-(6-Azidohexyl)-3-benzyloxypyridine-2-one (157e)

Reaction of 3-benzyloxypyridine-2-one (0.50 g, 2.49 mmol) and 156d (0.66g, 2.98 mmol) within 16 h as described for synthesis of 157a gavecompound 157e (0.48 g, 60%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃)δ 7.15 (m, 5H), 6.65 (dd, J=7.4, 1.8 Hz, 1H), 6.45 (dd, J=7.4, 1.8 Hz,1H), 5.81 (t, J=7.1 Hz, 1H), 4.92 (s, 2H), 3.76 (t, J=6.8 Hz, 2H), 3.05(t, J=6.8 Hz, 2H), 1.58 (m, 2H), 1.40 (m, 2H), 1.90 (m, 4H). ¹³C NMR(100 MHz, CDCl₃) δ 157.55, 148.25, 135.95, 128.60, 128.10, 126.95,126.50, 115.05, 104.90, 70.50, 51.05, 49.20, 28.65, 28.50, 26.20, 25.80.HRMS (FAB) calcd for C₁₈H₂₃N₄O₂ [M+H]⁺ 327.1821. found 327.1848.

1-(7-Azidoheptyl)-3-benzyloxypyridine-2-one (157f)

Reaction of 3-benzyloxypyridine-2-one (0.79 g, 3.38 mmol) and 156f (0.81g, 4.05 mmol) within 16 h as described for synthesis of 157a gavecompound 157f (0.55 g, 48%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃)δ 7.42 (dd, J=7.8, 1.0 Hz, 2H), 7.31 (m, 3H), 6.85 (dd, J=6.9, 1.7 Hz,1H), 6.61 (dd, J=7.4, 1.7 Hz, 1H), 5.99 (m, 1H), 5.09 (s, 2H), 3.93 (m,2H), 3.23 (t, J=6.9 Hz, 2H), 1.74 (m, 2H), 1.55 (m, 2H), 1.34 (m, 6H).¹³C NMR (100 MHz, CDCl₃) δ 158.32, 149.19, 136.63, 129.15, 128.74,128.13, 127.52, 115.66, 104.69, 94.66, 70.96, 51.62, 50.03, 29.22,28.98, 26.77, 26.72.

1-Phenyltriazolylethyl-3-methoxypyridine-2-one (158a)

Phenylacetylene (0.167 g, 1.63 mmol) and 157a (0.265 g, 1.36 mmol) weredissolved in anhydrous THF (10 mL) and stirred under argon at roomtemperature. Copper (I) iodide (0.011 g, 0.07 mmol) and Hunig's base(0.1 mL) were then added to the reaction mixture, and stirring continuedfor 4 h. The reaction mixture was diluted with CH₂Cl₂ (40 mL) and washedwith 1:4 NH₄OH/saturated NH₄Cl (3×30 mL) and saturated NH₄Cl (30 mL).The organic layer was dried over Na₂SO₄ and concentrated in vacuo. Thecrude product was triturated with hexanes to give 366 mg (91%) of whitesolid 158a. ¹H NMR (400 MHz, CDCl₃) δ 7.68 (s, 1H), 7.47 (d, J=7.5 Hz,2H), 7.11 (m, 3H), 6.43 (d, J=7.4 Hz, 1H), 6.35 (dd, J=6.8, 0.9 Hz, 1H),5.78 (t, J=7.2 Hz, 1H), 4.57 (t, J=5.7 Hz, 2H), 4.25 (t, J=5.6 Hz, 2H),3.53 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 157.70, 149.08, 147.21, 129.40,128.30, 128.08, 127.80, 125.00, 120.91, 113.02, 105.44, 55.15, 49.72,47.44. HRMS (EI) calcd for C₁₆H₁₆N₄O₂ [M]⁺ 296.1273. found 296.1268.

1-Phenyltriazolylpropyl-3-benzyloxypyridine-2-one (158b)

Reaction of phenylacetylene (0.127 g, 1.25 mmol) and 157b (0.295 g, 1.04mmol) within 4 h as described for synthesis of 158a gave compound 158b(0.31 g, 77%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.00 (s, 1H),7.77 (d, J=7.3 Hz, 2H), 7.27 (m, 8H), 6.88 (d, J=5.7 Hz, 1H), 6.57 (d,J=6.4 Hz, 1H), 5.95 (t, J=7.1 Hz, 1H), 4.99 (s, 2H), 4.34 (t, J=6.4 Hz,2H), 3.94 (t, J=6.4 Hz, 2H), 2.32 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ157.92, 148.47, 147.30, 135.73, 130.26, 128.81, 128.49, 128.22, 127.76,127.71, 127.01, 125.33, 120.16, 114.95, 104.83, 70.33, 47.00, 46.48,29.30. HRMS (EI) calcd for C₂₃H₂₂N₄O₂ [M]⁺ 386.1743. found 386.1734.

1-Phenyltriazolylbutyl-3-benzyloxypyridine-2-one (158c)

Reaction of phenylacetylene (0.124 g, 1.21 mmol) and 157c (0.30 g, 1.01mmol) within 4 h as described for synthesis of 158a gave compound 158c(0.31 g, 77%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.83 (s, 1H),7.76 (m, 2H), 7.26 (m, 8H), 6.76 (dd, J=6.9, 1.5 Hz, 1H), 6.56 (dd,J=7.4, 1.5 Hz, 1H), 5.91 (t, J=7.1 Hz, 1H), 4.99 (s, 2H), 4.31 (m, 2H),3.89 (t, J=7.1 Hz, 2H), 1.85 (m, 2H), 1.67 (m, 2H). ¹³C NMR (100 MHz,CDCl₃) δ 157.71, 148.38, 147.20, 135.83, 130.29, 128.45, 128.41, 128.16,127.68, 127.63, 126.96, 125.25, 119.87, 114.99, 104.60, 70.26, 49.11,48.02, 26.78, 25.65. HRMS (EI) calcd for C₂₄H₂₄N₄O₂ [M]⁺ 400.1899. found400.1888.

1-Phenyltriazolylpentyl-3-benzyloxypyridine-2-one (158d)

Reaction of phenylacetylene (0.04 g, 0.32 mmol) and 157d (0.10 g, 0.32mmol) within 4 h as described for synthesis of 158a gave compound 158d(0.092 g, 72%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.78 (m, 3H),7.30 (m, 8H), 6.79 (d, J=6.8 Hz, 1H), 6.57 (d, J=7.3 Hz, 1H), 5.94 (t,J=7.1 Hz, 1H), 5.02 (s, 2H), 4.31 (t, J=7.0 Hz, 2H), 3.87 (t, J=7.2 Hz,2H), 1.91 (m, 2H), 1.74 (m, 2H), 1.30 (m, 2H). ¹³C NMR (100 MHz, CDCl₃)δ 157.80, 148.57, 147.39, 136.02, 130.44, 128.64, 128.56, 128.27,127.81, 127.71, 127.05, 125.40, 119.57, 115.12, 104.49, 70.40, 49.71,49.06, 29.42, 28.03, 23.05.

1-Phenyltriazolylhexyl-3-benzyloxypyridine-2-one (158e)

Reaction of phenylacetylene (0.80 g, 0.78 mmol) and 157e (0.21 g, 0.65mmol) within 4 h as described for synthesis of 158a gave compound 158e(0.16 g, 56%) as a white solid. NMR (400 MHz, CDCl₃) δ 7.66 (m, 3H),7.20 (m, 8H), 6.68 (dd, J=6.9, 1.5 Hz, 1H), 6.46 (dd, J=7.4, 1.5 Hz,1H), 5.82 (t, J=7.1 Hz, 1H), 4.92 (s, 2H), 4.18 (t, J=6.8 Hz, 2H), 3.75(t, J=6.8 Hz, 2H), 1.76 (m, 2H), 1.56 (m, 2H), 1.19 (m, 4H). ¹³C NMR(100 MHz, CDCl₃) δ 157.64, 148.46, 147.27, 135.95, 130.36, 128.57,128.48, 128.19, 127.72, 127.60, 126.96, 125.32, 119.37, 115.02, 104.3770.45, 50.03, 49.35, 29.90, 28.66, 25.87, 25.78. HRMS (FAB) calcd forC₂₆H₂₉N₄O₂ [M+H]⁺ 429.2290. found 429.2291.

1-Phenyltriazolylheptyl-3-benzyloxypyridine-2-one (158f)

Reaction of phenylacetylene (0.90 g, 0.88 mmol) and 157f (0.25 g, 0.735mmol) within 4 h as described for synthesis of 158a gave compound 1581(0.23 g, 63%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.79 (m, 3H),7.31 (m, 8H), 6.83 (dd, J=6.9, 1.7 Hz, 1H), 6.60 (dd, J=7.4, 1.7 Hz,1H), 5.97 (t, J=7.1 Hz, 1H), 5.07 (s, 2H), 4.35 (t, J=7.1 Hz, 2H), 3.91(m, 2H), 1.88 (m, 2H), 1.71 (m, 2H), 1.32 (d, J=10.0 Hz, 6H). ¹³C NMR(100 MHz, CDCl₃) δ 158.31, 149.13, 147.92, 136.57, 130.93, 129.15,129.04, 128.74, 128.28, 128.15, 127.51, 125.90, 125.86, 119.75, 115.60,104.79, 70.92, 50.52, 49.90, 30.42, 29.13, 28.72, 26.53, 26.46. HRMS(FAB) calcd for C₂₇H₃₁N₄O₂ [M+H]⁺ 443.2447. found 443.2494.

1-Phenyltriazolylethyl-3-hydroxypyridine-2-one (159a)

To a solution of 158a (0.10 g, 0.338 mmol) in dry CH₂Cl₂ (8 mL) wasslowly added 1M BBr₃ (1.2 equiv) at −30° C. under argon atmosphere. Thereaction mixture was stirred for 48 h at room temperature. The mixturewas again cooled to −30° C. and the MeOH (5 mL) was slowly added to themixture. After evaporation of solvent, the residue was adjusted to pH 7with 1M NaOH and then extracted with CHCl₃ (30 mL×3). The combinedorganic layer dried over Na₂SO₄ to give 79 mg (83%) of 159a as slightlybrownish solid without any further purification required. ¹H NMR (400MHz, DMSO) δ 9.11 (s, 1H), 8.51 (s, 1H), 7.78 (d, J=7.3 Hz, 2H), 7.43(t, J=7.6 Hz, 2H), 7.31 (t, J=7.3 Hz, 1H), 6.75 (d, J=5.9 Hz, 1H), 6.64(d, J=6.0 Hz, 1H), 5.95 (t, J=7.0 Hz, 1H), 4.76 (t, J=5.5 Hz, 2H), 4.42(t, J=5.6 Hz, 2H). ¹³C NMR (100 MHz, DMSO) δ 158.25, 147.17, 146.77,131.11, 129.34, 128.41, 128.31, 125.54, 122.23, 115.38, 105.76, 49.42,48.33. HRMS (EI) calcd for C₁₅H₁₄N₄O₂ [M]⁺ 282.1117. found 282.1120.

1-Phenyltriazolylpropyl-3-hydroxypyridine-2-one (159b)

To a solution of 158b (0.29 g, 1.01 mmol) in CH₂Cl₂:EtOAc:MeOH (2:2:1,10 mL) was added 10% Pd on carbon (15 mg). Reaction was stirred underballoon hydrogen pressure for 5 h. Reaction mixture was filtered andsolvent was evaporated. Column chromatography using gradientCH₂Cl₂:Acetone (max 20%) gave pure 159b (0.19 g, 64%) as a slightlybrownish solid. ¹H NMR (400 MHz, DMSO) δ 9.03 (s, 1H), 8.62 (s, 1H),7.82 (d, J=7.6 Hz, 2H), 7.38 (m, 3H), 7.15 (d, J=6.6 Hz, 1H), 6.68 (d,J=6.4 Hz, 1H), 6.11 (t, J=6.7 Hz, 1H), 4.43 (t, J=6.4 Hz, 2H), 3.99 (m,2H), 2.27 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 147.49, 129.71, 128.52,127.99, 125.29, 120.42, 107.98, 66.61, 47.10, 29.27. (expected aromaticcarbons=11; quaternary carbons can't be seen even after repeated cycles)HRMS (EI) calcd for C₁₆H₁₆N₄O₂ [M]⁺ 296.1273. found 296.1274.

1-Phenyltriazolylbutyl-3-hydroxypyridine-2-one (159c)

Reaction of 158c (0.25 g, 0.62 mmol) in anhydrous THF as described forsynthesis of 159b gave 159c (0.12 g, 63%) of pure product. ¹H NMR (400MHz, CDCl₃) δ 7.79 (m, 3H), 7.41 (t, J=7.6 Hz, 2H), 7.32 (t, J=7.4 Hz,1H), 6.76 (m, 3H), 6.13 (t, J=7.2 Hz, 1H), 4.45 (t, J=6.8 Hz, 2H), 4.02(t, J=7.1 Hz, 2H), 1.99 (m, 2H), 1.82 (m, 2H). ¹³C NMR (100 MHz, CDCl₃)δ 158.47, 147.63, 146.62, 130.41, 128.69, 127.99, 126.60, 125.53,119.80, 114.15, 107.10, 49.38, 48.57, 27.02, 26.01. HRMS (EI) calcd forC₁₇H₁₈N₄O₂ [M]⁺ 310.1430. found 310.1425.

1-Phenyltriazolylpentyl-3-hydroxypyridine-2-one (159d)

Reaction of 158d (0.085 g, 0.20 mmol) in anhydrous THF as described forsynthesis of 159b gave 159d (0.045 g, 68%) of pure product. ¹H NMR (400MHz, DMSO) δ 8.56 (s, 1H), 7.82 (d, J=8.1 Hz, 2H), 7.43 (t, J=7.7 Hz,2H), 7.32 (t, J=7.4 Hz, 1H), 7.10 (dd, J=6.8, 1.6 Hz, 1H), 6.65 (dd,J=7.2, 1.5 Hz, 1H), 6.05 (1, J=7.0 Hz, 1H), 4.38 (t, J=7.0 Hz, 2H), 3.89(t, J=7.2 Hz, 2H), 1.89 (m, 2H), 1.67 (m, 2H), 1.25 (m, 2H). ¹³C NMR(100 MHz, CDCl₃) δ 158.44, 147.67, 146.61, 130.51, 128.72, 128.01,126.71, 125.57, 119.54, 113.81, 106.86, 49.86, 49.38, 29.58, 28.31,23.19. HRMS (FAB) calcd for C₁₈H₂₁N₄O₂ [M+H]⁺ 325.1664. found 325.1683.

1-Phenyltriazolylhexyl-3-hydroxypyridine-2-one (159e)

Reaction of 158e (0.15 g, 0.35 mmol) in anhydrous THF as described forsynthesis of 159b gave 159e (0.081 g, 69%) of pure product. ¹H NMR (400MHz, CDCl₃) δ 7.81 (m, 3H), 7.42 (m, 2H), 7.32 (m, 1H), 6.77 (m, 2H),6.12 (t, J=7.1 Hz, 1H), 4.38 (t, J=7.1 Hz, 2H), 3.95 (m, 2H), 1.95 (m,2H), 1.76 (d, J=7.0 Hz, 2H), 1.39 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) δ158.20, 147.25, 146.40, 130.30, 128.60, 127.85, 126.65, 125.40, 119.65,113.90, 106.80, 50.20, 49.60, 30.05, 28.95, 26.00, 25.90. HRMS (FAB)calcd for C₁₉H₂₃N₄O₂ [M+H]⁺ 339.1821. found 339.1814.

1-Phenyltriazolylheptyl-3-hydroxypyridine-2-one (159f)

Reaction of 158f (0.22 g, 0.50 mmol) in anhydrous THF as described forsynthesis of 159b gave 159f (0.13 g, 74%) of pure product. ¹H NMR (400MHz, DMSO) δ 8.91 (s, 1H), 8.57 (s, 1H), 7.82 (d, J=7.4 Hz, 2H), 7.42(t, J=7.6 Hz, 2H), 7.31 (t, J=7.3 Hz, 1H), 7.09 (d, J=6.6 Hz, 1H), 6.64(d, J=7.0 Hz, 1H), 6.04 (t, J=6.9 Hz, 1H), 4.36 (t, J=7.0 Hz, 2H), 3.86(t, J=7.2 Hz, 2H), 1.83 (m, 2H), 1.61 (m, 2H), 1.26 (m, 6H). ¹³C NMR(100 MHz, CDCl₃) δ 158.80, 147.89, 147.00, 130.88, 129.05, 128.30,127.12, 125.87, 119.83, 114.13, 107.10, 50.48, 50.05, 30.37, 29.20,28.67, 26.46, 26.42. HRMS (FAB) calcd for C₂₀H₂₅N₄O₂ [M+H]⁺ 353.1977.found 353.1992.

1-Phenyltriazolylethyl-3-methoxypyridine-2-thione (160)

To a stirring solution of Lawesson's reagent (0.075 g, 0.185 mmol) intoluene (10 mL), was added starting material 158a (0.10 g, 0.33 mmol).Reaction mixture was heated to reflux temperature. After overnightreaction, solvent was evaporated under reduced pressure. Crude productwas dissolved in small amount of CH₂Cl₂ to load onto preparative-TLC andeluted with CHCl₃:Acetone:EtOH (10:1:0.2) to give pure yellow solid 160(0.07 g, 68%). ¹H NMR (400 MHz, CDCl₃) δ 7.69 (d, J=7.4 Hz, 2H), 7.56(s, 1H), 7.35 (t, J=7.5 Hz, 2H), 7.28 (m, 1H), 6.97 (d, J=6.6 Hz, 1H),6.62 (d, J=7.8 Hz, 1H), 6.37 (m, 1H), 5.13 (t, J=5.6 Hz, 2H), 5.03 (t,J=5.6 Hz, 2H), 3.86 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 171.92, 158.94,147.52, 132.68, 129.92, 128.69, 128.14, 125.43, 120.96, 111.72, 110.22,56.72, 56.63, 46.38. HRMS (EI) calcd for C₁₆H₁₆N₄OS [M]⁺ 312.104. found312.1039.

1-Phenyltriazolylethyl-3-hydroxypyridine-2-thione (161)

Reaction of 160 (0.062 g, 0.198 mmol) and 1M BBr₃ in CH₂Cl₂ (0.218 mmol)within 48 h as described for synthesis of 148a gave compound 161 (0.055g, 93%) as a dark violet solid. ¹H NMR (400 MHz, CDCl₃) δ 8.40 (s, 1H),7.73 (d, J=7.3 Hz, 2H), 7.51 (s, 1H), 7.40 (t, J=7.5 Hz, 2H), 7.33 (t,J=7.3 Hz, 1H), 6.95 (dd, J=9.2, 7.2 Hz, 2H), 6.46 (m, 1H), 5.08 (m, 4H).¹³C NMR (100 MHz, CDCl₃) δ 168.47, 155.10, 147.75, 132.00, 129.82,128.77, 128.30, 125.53, 120.90, 113.66, 112.61, 57.45, 46.41. HRMS (EI)calcd for C₁₅H₁₄N₄OS [M]⁺ 298.088. found 298.0888.

1-Phenyltriazolylpropyl-3-hydroxypyridine-2-thione (162b)

159b (0.062 g, 0.21 mmol) was ground together with P₄S₁₀ (0.047 g, 0.105mmol) in mortar and pestle to form grey powder. The powder was stirredunder argon in a flask fitted with condenser and heated to 175° C. for 2h. The reaction flask was covered with aluminum foil during reaction.After 2 h of reaction, flask was cooled down to room temperature. 25 mLof CH₂Cl₂:MeOH (10%) mixtures was added and stirred for 15 min. Thisreaction mixture then washed with water (2×30 mL). Organic layer driedwith Na2SO4 and evaporated to yield crude product which was purified byprep-TLC (Eluent —CH₂Cl₂:MeOH (8%) to give 162b (0.04 g, 60%) as olivegreen solid. ¹H NMR (400 MHz, CDCl₃) δ 8.47 (s, 1H), 7.82 (m, 3H), 7.50(d, J=6.5 Hz, 1H), 7.43 (t, J=7.5 Hz, 2H), 7.34 (t, J=7.4 Hz, 1H), 6.97(m, 1H), 6.66 (m, 1H), 4.62 (t, J=6.9 Hz, 2H), 4.48 (m, 2H). ¹³C NMR(100 MHz, CDCl₃) δ 168.59, 155.22, 148.03, 131.85, 130.17, 128.84,128.29, 125.61, 119.84, 113.87, 112.28, 54.99, 46.95, 28.02. HRMS (EI)calcd for C₁₆H₁₆N₄OS [M]⁺ 312.104. found 312.1060.

1-Phenyltriazolylbutyl-3-hydroxypyridine-2-thione (162c)

Reaction of 159c (0.055 g, 0.18 mmol) and P₄S₁₀ (0.051 g, 0.115 mmol)under neat conditions as described for synthesis of 162b gave compound162c (0.032 g, 55%) as a olive green solid. δ 8.50 (s, 1H), 7.81 (m,3H), 7.36 (m, 4H), 6.94 (d, J=7.6 Hz, 1H), 6.62 (t, J=6.8 Hz, 1H), 4.54(t, J=7.6 Hz, 2H), 4.46 (t, J=6.4 Hz, 2H), 2.03 (m, 4H). ¹³C NMR (100MHz, CDCl₃) δ 168.51, 155.08, 147.73, 131.02, 130.36, 128.79, 128.13,125.58, 119.91, 113.87, 112.10, 56.96, 49.41, 27.02, 25.08. HRMS (EI)calcd for C₁₇H₁₈N₄OS [M]⁺ 326.1201. found 326.1200.

1-Phenyltriazolylpentyl-3-hydroxypyridine-2-thione (162d)

Reaction of 159d (0.025 g, 0.077 mmol) and P₄S₁₀ (0.020 g, 0.046 mmol)under neat conditions as described for synthesis of 162b gave compound162d (0.019 g, 73%) as a olive green solid. ¹H NMR (400 MHz, DMSO) δ8.55 (d, J=2.9 Hz, 2H), 7.83 (t, J=7.8 Hz, 3H), 7.43 (q, J=7.4 Hz, 2H),7.32 (t, J=6.6 Hz, 1H), 7.08 (m, 1H), 6.92 (dd, J=31.0, 6.8 Hz, 1H),6.81 (m, 1H), 4.51 (m, 2H), 4.41 (t, J=7.0 Hz, 2H), 1.89 (m, 4H), 1.32(m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 168.37, 155.10, 147.66, 131.04,130.46, 128.75, 128.05, 125.57, 119.68, 113.69, 111.96, 57.73, 49.73,29.47, 27.12, 23.04. HRMS (EI) calcd for C₁₈H₂₀N₄OS [M]⁺ 340.135. found326.1364.

1-Phenyltriazolylhexyl-3-hydroxypyridine-2-thione (162e)

Reaction of 159e (0.18 g, 0.532 mmol) and P₄S₁₀ (0.118 g, 0.266 mmol)under neat conditions as described for synthesis of 162b gave compound162e (0.080 g, 42%) as a olive green semi-solid. ¹H NMR (400 MHz, CDCl₃)δ 8.57 (s, 1H), 7.83 (d, J=7.5 Hz, 2H), 7.76 (s, 1H), 7.41 (t, J=7.5 Hz,2H), 7.32 (m, 2H), 6.94 (m, 1H), 6.72 (m, 1H), 4.49 (m, 2H), 4.40 (t,J=6.9 Hz, 2H), 1.97 (m, 4H), 1.36 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) δ164.38, 162.90, 147.70, 130.54, 128.76, 128.03, 126.64, 125.61, 119.51,118.75, 118.29, 59.74, 50.10, 29.91, 27.99, 25.82, 25.65. HRMS (ESI)calcd for C₁₉H₂₃N₄OS [M+H]⁺ 355.158. found 355.1632.

1-Phenyltriazolylheptyl-3-hydroxypyridine-2-thione (162f)

Reaction of 159f (0.12 g, 0.532 mmol) and P₄S₁₀ (0.076 g, 0.17 mmol)under neat conditions as described for synthesis of 162b gave compound162f (0.054 g, 43%) as a olive green semi-solid. ¹H NMR (400 MHz, DMSO)δ 8.55 (m, 1H), 7.81 (d, J=8.2 Hz, 2H), 7.65 (d, J=6.2 Hz, 1H), 7.42 (m,2H), 7.30 (m, 1H), 7.02 (m, 1H), 6.85 (m, 1H), 4.50 (m, 2H), 4.36 (t,J=7.1 Hz, 2H), 1.85 (d, J=7.0 Hz, 4H), 1.33 (s, 6H). ¹³C NMR (100 MHz,CDCl₃) δ 147.76, 130.66, 128.76, 130.39, 128.98, 128.80, 128.06, 125.97,125.67, 119.46, 60.10, 50.25, 30.11, 29.67, 28.33, 26.13, 26.10. HRMS(FAB) calcd for C₂₀H₂₅N₄OS [M+H]⁺ 369.174. found 369.1762.

Example 10 3^(rd) Generation 3-Hydroxypyridine-2-thione-based HDACInhibitors

A 3^(rd) generation of HDAC inhibitors containing 5-carbon methylenespacers were prepared. The synthetic route to the 3^(rd) generationcompounds was identical to that described for the 2^(nd) generationcompounds. The new surface cap recognition cap groups were coupled toZBG modified linker using Cu(I)-catalyzed Huisgen cycloaddition to giveintermediates in good to excellent yields. As described earlier,Lawessons' chemistry followed by deprotection with BBr₃ resulted indesired compounds 166a-166g and 167a-168g. Carbonyl derivatives werealso synthesized using similar deprotection chemistry (Scheme 21).

The 3^(rd) generation compounds in 3-HPT series were tested againstHDAC1, HDAC6 and HDAC8 using the label-free SAMDI mass spectrometryassay as described above. The trend in HDAC inhibition activity was verysimilar to that observed in the early generation compounds with thethione compounds showing superior activity compared to the carbonylderivatives. For HDAC8, the results from this study indicate thatsubstitution on the phenyl ring as cap group is not helpful foranti-HDAC activity. Almost all of the cap groups other than simplephenyl ring failed to improve compound potency (compounds 166a-166g;167a-167g in table 13). However, for HDAC6 docking substitution patternon phenyl ring yielded improved potency. In case of methyl substitution,para and ortho substitution resulted in more potent drugs than metasubstitution. This trend was very similar to 1^(st) generation methylsubstituted compounds (comparing compound 149d-149f and 167a-167e). Outof para (compound 167d) and meta (compound 167e) cyano substitution,meta showed superior activity compared to para substitution (compound167e, IC₅₀=356 nM against HDAC6). In addition, substitution withelectron donating group N,N-dimethylamino and electron withdrawing groupsuch as shown in compound 167g did not surpass the activity of compound167e. In order to gain additional insight into binding interactions of2^(nd) and 3^(rd) generation of compounds at the enzyme active site,docking analyses were performed on the most active compounds in thisseries against HDAC8.

TABLE 13 In vitro HDAC Inhibition of 3^(rd) Generation Compounds IC₅₀(nM) COMPOUNDS HDAC1 HDAC6 HDAC8

  166a 66% NI 15%

  167a 31% 807 ± 207 2533 +/− 823 

  166b 88% NI NI

  167b 19% 1100 ± 443  1660 +/− 416 

  166c NT NI  4%

  167c NT 637 ± 160 2402 +/− 263 

  166d 70% NI 26%

  167d 33% 905 ± 249 1465 +/− 217 

  166e 71% NI NI

  167e ND 356 ± 72  2831 +/− 520 

  166f 59% NI 28%

  167f 28% 1006 ± 425  1482 +/− 389 

  166g 71% NI 14%

  167g 55% 661 ± 121 2258 + /− 1005 SAHA 38 +/− 2 95% 232 ± 19  SAHA isused for a positive control; NI — No significant Inhibition (below 20%Inhibition); % inhibitions of the compounds at 10 μM are given if theIC₅₀ was above 10 μM.

Docking of 3^(rd) Generation Compounds

Compound 162d adopts a docked pose at HDAC8 active site which presentsthe phenyl cap group into a deep, non-solvent exposed hydrophobicpocket, while the 5-carbon methylene spacer optimally fills theconnecting the hydrophobic pocket to the Zn²⁺ active site. The idealspacer length of the 5-carbon methylene group facilitates a properpresentation off the 3-HPT ZBG to the active site Zn²⁺. The deep pocketwhere the phenyl ring orients consists of Tyr111, Ala112, Leu155, Tyr154AA residues. Two tyrosine residues are within 3.5 Å of the phenyl capgroup of compound 162d and are ideally suited for pi-stackinginteraction. Tyr154 is expected to have stronger pi-stacking interactionsince plane of its ring is parallel to the phenyl cap group of compound162d. However, Tyr111 will contribute less to the pi-stackinginteraction because the edge of its phenol ring is oriented toward theplane of the cap group 162d. Other AA residues such as Leu115 and Ala112are within 3.5 Å and contribute to the overall hydrophobicity of thepocket into which the phenyl cap group of compound 162d is bound. Thismight lead to lower binding energy conformation and hence better IC₅₀value for this 2^(nd) generation compound.

The docked structures of 162d and six methylene linker compound 162ewere overlayed. Both compounds bind to the same pocket and adopt almostthe similar orientation, except for slight kinks, at the methylenegroups. The major difference between the docked poses of 162d and 162eis at their phenyl cap group which is slightly pushed outside ofpi-stacking interaction with Tyr154 in compound 162e and thus abolishingthis important interaction. This could be the reason that there isalmost 7-fold activity difference between 162d and 162e.

Similarly, compound 167d binds to same pocket as discussed earlier.However para-cyano substitution on cap group 167d causes its phenyl ringto be pushed out of the binding pocket seen for compound 162d (Figure20). Though two tyrosine residues are not far away from the phenyl capgroup (˜3.2 Å), their orientation is not optimum for pi-stackinginteraction with the phenyl group of 167d. Moreover, hydrophobicinteractions with Ala112 and Leu155 are completely lost. However, thereis a compensatory pi-stacking interaction with Trp141 which may explainthe better activity for compound 167d relative to 162e.

The preference for the HDAC8 isoform over HDAC1 was consistent foralmost all of the compounds tested in this series. This is an importantand interesting observation considering the fact that other aromatic ZBGsuch as benzamide showed HDAC1 selectivity (250 fold). To obtaininformation on the structural basis of the observed disparity in theHDAC isoform selectivity, molecular docking analysis was performed onlead compound 149d with two HDAC isoforms. However, HDAC1 docking of149d revealed that there was no fruitful interaction of compound 149d atHDAC1 enzyme active site. Repeated docking suggested that the ZBG of149d was unable to deeply penetrate (˜14 Å) the Zn² active site ofHDAC1.

Synthetic Procedures1-(4-Tolyl)triazolylpentyl-3-methoxypyridine-2-thione (165a)

Reaction of 164a (0.235 g, 0.667 mmol) and Lawesson's reagent (0.162 g,0.40 mmol) in toluene (12 mL) within 12 h as described for the synthesisof 147a gave compound 165a (0.232 g, 95%) as yellow solid. ¹H NMR (400MHz, CDCl₃) δ 7.77 (s, 1H), 7.73 (d, J=8.2 Hz, 2H), 7.33 (m, 1H), 7.25(dd, J=11.4, 5.2 Hz, 2H), 6.66 (d, J=7.8 Hz, 1H), 6.59 (dd, J=7.8, 6.5Hz, 1H), 4.60 (m, 2H), 4.43 (t, J=6.9 Hz, 2H), 3.91 (s, 3H), 2.38 (s,3H), 2.01 (m, 4H), 1.43 (m, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 171.03,158.50, 147.11, 137.37, 131.77, 129.02, 127.33, 125.03, 119.32, 111.60,109.78, 56.27, 49.28, 30.48, 29.05, 26.51, 22.57, 20.82. HRMS (EI) calcdfor C₂₀H₂₄N₄OS [M]⁺ 368.167. found 368.1667.

1-(3-Tolyl)triazolylpentyl-3-methoxypyridine-2-thione (165b)

Reaction of 164b (0.137 g, 0.389 mmol) and Lawesson's reagent (0.094 g,0.223 mmol) in toluene (10 mL) within 12 h as described for thesynthesis of 147a gave compound 165b (0.104 g, 73%) as yellow solid. ¹HNMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.62 (s, 1H), 7.54 (d, J=7.7 Hz,1H), 7.26 (m, 2H), 7.07 (d, J=7.6 Hz, 1H), 6.59 (m, 1H), 6.52 (dd,J=7.8, 6.5 Hz, 1H), 4.50 (m, 2H), 4.34 (t, J=7.0 Hz, 2H), 3.81 (s, 3H),2.32 (s, 3H), 1.90 (m, 4H), 1.33 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ171.47, 158.81, 147.45, 138.19, 131.83, 130.21, 128.58, 128.46, 126.03,122.48, 119.68, 111.72, 109.80, 56.55, 56.47, 49.50, 29.24, 26.70,22.77, 21.18. HRMS (EI) calcd for C₂₀H₂₄N₄OS [M]⁺ 368.1671. found368.1682.

1-(2-Tolyl)triazolylpentyl-3-methoxypyridine-2-thione (165c)

Reaction of 164c (0.124 g, 0.351 mmol) and Lawesson's reagent (0.085 g,0.21 mmol) in toluene (10 mL) within 12 h as described for the synthesisof 147a gave compound 165c (0.093 g, 72%) as yellow solid. ¹H NMR (400MHz, CDCl₃) δ 7.72 (m, 1H), 7.27 (m, 2H), 6.60 (m, 1H), 4.57 (m, 1H),4.42 (t, J=7.0 Hz, 1H), 3.86 (s, 1H), 2.44 (s, 1H), 1.99 (m, 2H), 1.41(dt, J=15.2, 7.6 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 171.81, 159.04,146.84, 135.33, 131.86, 130.74, 129.81, 128.68, 127.94, 125.93, 121.86,111.78, 109.79, 104.88, 56.75, 56.61, 49.60, 29.45, 26.85, 22.97, 21.36.HRMS (EI) calcd for C₂₀H₂₄N₄OS [M]⁺ 368.167. found 368.1662.

1-(4-Benzonitrile)triazolylpentyl-3-methoxypyridine-2-thione (165d)

Reaction of 164d (0.210 g, 0.551 mmol) and Lawesson's reagent (0.133 g,0.330 mmol) in toluene (12 mL) within 12 h as described for thesynthesis of 147a gave compound 165d (0.146 g, 67%) as yellow solid. ¹HNMR (400 MHz, CDCl₃) δ 7.64 (m, 3H), 7.28 (m, 1H), 6.73 (d, J=8.3 Hz,2H), 6.61 (d, J=7.6 Hz, 1H), 6.52 (t, J=7.1 Hz, 1H), 4.50 (m, 2H), 4.31(t, J=6.7 Hz, 2H), 3.81 (s, 3H), 2.92 (s, 6H), 1.89 (m, 4H), 1.32 (m,2H); ¹³C NMR (100 MHz, CDCl₃) δ 171.44, 158.81, 149.78, 147.77, 131.91,126.37, 119.05, 118.34, 112.52, 111.73, 109.84, 56.60, 56.49, 49.44,40.45, 29.29, 26.74, 22.81. HRMS (EI) calcd for C₂₁H₂₇N₅OS [M]⁺ 397.193.found 397.1928.

1-(3-Benzonitrile)triazolylpentyl-3-methoxypyridine-2-thione (165e)

Reaction of 164e (0.190 g, 0.523 mmol) and Lawesson's reagent (0.130 g,0.314 mmol) in toluene (10 mL) within 12 h as described for thesynthesis of 147a gave compound 165e (0.133 g, 67%) as yellow solid. ¹HNMR (400 MHz, CDCl₃) δ 7.98 (s, 1H), 7.84 (d, J=8.4 Hz, 2H), 7.56 (d,J=8.4 Hz, 2H), 7.30 (d, J=6.5 Hz, 1H), 6.60 (d, J=7.0 Hz, 1H), 6.52 (dd,J=7.7, 6.6 Hz, 1H), 4.48 (m, 2H), 4.34 (t, J=6.9 Hz, 2H), 3.76 (s, 3H),1.88 (m, 4H), 1.31 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 171.23, 158.70,145.36, 134.76, 132.30, 131.78, 125.68, 121.20, 118.49, 111.83, 110.73,109.92, 56.43, 49.50, 29.01, 26.56, 22.56. HRMS (EI) calcd forC₂₀H₂₁N₅OS [M]⁺ 379.146. found 379.1466.

1-(4-(N,N-dimethylaniline))triazolylpentyl-3-methoxypyridine-2-thione(165f)

Reaction of 1641 (0.145 g, 0.40 mmol) and Lawesson's reagent (0.098 g,0.241 mmol) in toluene (10 mL) within 12 h as described for thesynthesis of 147a gave compound 1651 (0.149 g, 98%) as yellow solid. ¹HNMR (400 MHz, CDCl₃) δ 8.12 (t, J=1.4 Hz, 1H), 8.06 (m, 1H), 7.90 (s,1H), 7.59 (dt, J=7.7, 1.4 Hz, 1H), 7.52 (t, J=7.8 Hz, 1H), 7.33 (dd,J=6.5, 1.4 Hz, 1H), 6.67 (dd, 7.9, 1.3 Hz, 1H), 6.60 (dd, J=7.8, 6.5 Hz,1H), 4.59 (m, 2H), 4.46 (t, J=6.9 Hz, 2H), 3.89 (s, 3H), 2.02 (m, 4H),1.43 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 171.30, 158.73, 145,13, 131.82,131.71, 130.97, 129.52, 129.44, 128.64, 120.61, 118.28, 112.49, 111.81,109.91, 56.44, 49.62, 29.11, 26.64, 22.68. HRMS (EI) calcd forC₂₀H₂₁N₅OS [M]⁺ 379.146. found 379.1469.

1-(4-(3-Trifluoromethyl)-benzonitrile)triazolylpentyl-3-methoxypyridine-2-thione(165g)

Reaction of 164g (0.090 g, 0.2088 mmol) and Lawesson's reagent (0.051 g,0.1252 mmol) in toluene (10 mL) within 12 h as described for thesynthesis of 147a gave compound 165g (0.075 g, 81%) as yellow solid. ¹HNMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 8.12 (d, J=6.7 Hz, 2H), 7.86 (d,J=8.1 Hz, 1H), 7.36 (d, J=6.5 Hz, 1H), 6.68 (d, J=7.8 Hz, 1H), 6.61 (t,J=7.2 Hz, 1H), 4.59 (m, 2H), 4.47 (m, 2H), 3.88 (s, 3H), 2.02 (m, 4H),1.43 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 171.84, 159.14, 144.54, 135.55,135.22, 133.51, 133.18, 131.81, 128.59, 121.86, 120.86, 115.46, 112.00,109.97, 108.47, 77.31, 76.99, 76.67, 56.69, 49.87, 29.19, 26.77, 22.77.HRMS (EI) calcd for C₂₁H₂₀N₅OSF₃ [M]⁺ 447.134. found 447.1341.

1-(4-Tolyl)triazolylpentyl-3-hydroxypyridine-2-one (166a)

Reaction of 164a (0.10 g, 0.284 mmol) and 1M BBr₃ (0.34 mL) in CH₂Cl₂ (8mL) within 48 h as described for the synthesis of 148a gave compound166a (0.049 g, 52%) as light brown solid. ¹H NMR (400 MHz, CDCl₃) δ 7.71(m, 3H), 7.24 (m, 2H), 6.76 (t, J=6.4 Hz, 2H), 6.12 (t, J=7.0 Hz, 1H),4.40 (t, J=6.9 Hz, 2H), 3.96 (t, J=7.1 Hz, 2H), 2.38 (s, 3H), 2.00 (m,2H), 1.82 (m, 2H), 1.39 (m, 2H); ¹³C NMR (100 MHz, DMSO) δ 171.80,146.31, 137.02, 129.40, 128.08, 125.02, 120.79, 114.36, 105.20, 49.27,48.22, 29.17, 28.01, 22.85, 20.82. HRMS (EI) calcd for C₁₉H₂₂N₄O₂ [M]⁺338.1743. found 338.1750.

1-(3-Tolyl)triazolylpentyl-3-hydroxypyridine-2-one (166b)

Reaction of 164b (0.073 g, 0.207 mmol) and 1M BBr₃ (0.24 mL) in CH₂Cl₂(8 mL) within 48 h as described for the synthesis of 148a gave compound166b (52 mg, 74%) as light brown solid. ¹H NMR (400 MHz, CDCl₃) δ 7.74(s, 1H), 7.67 (s, 1H), 7.59 (d, J=7.7 Hz, 1H), 7.30 (t, J=7.6 Hz, 1H),7.14 (d, J=7.4 Hz, 1H), 6.76 (t, J=6.2 Hz, 2H), 6.12 (t, J=7.1 Hz, 1H),4.39 (t, J=6.9 Hz, 2H), 3.96 (t, J=7.2 Hz, 2H), 2.39 (s, 3H), 1.99 (m,2H), 1.80 (m, 2H), 1.38 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 158.58,147.87, 146.52, 138.46, 130.38, 128.86, 128.67, 126.75, 126.31, 122.72,119.48, 113.61, 106.85, 49.91, 49.45, 29.65, 28.38, 23.27, 21.38. HRMS(EI) calcd for C₁₉H₂₂N₄O₂ [M]⁺ 338.174. found 338.1741.

1-(2-Tolyl)triazolylpentyl-3-hydroxypyridine-2-one (166c)

Reaction of 164c (0.065 g, 0.18 mmol) and 1M BBr₃ (0.27 mL) in CH₂Cl₂ (8mL) within 48 h as described for the synthesis of 148a gave compound166c (54 mg, 87%) as light brown solid. ¹H NMR (400 MHz, dorso) δ 8.92(s, 1H), 8.36 (s, 1H), 7.71 (m, 1H), 7.25 (m, 1H), 7.09 (d, J=6.8 Hz,1H), 6.64 (d, J=6.9 Hz, 1H), 6.04 (t, J=7.0 Hz, 1H), 4.39 (t, J=6.9 Hz,1H), 3.88 (t, J=7.0 Hz, 1H), 2.41 (s, 1H), 1.90 (m, 1H), 1.67 (m, 1H),1.25 (m, 1H). ¹³C NMR (100 MHz, CD₃OD) δ 146.28, 135.00, 130.15, 128.96,128.21, 127.72, 125.38, 122.09, 106.72, 49.43, 48.98, 29.05, 27.78,22.69, 20.09. HRMS (EI) calcd for C₁₉H₂₂N₄O₂ [M]⁺ 338.174. found338.1741.

1-(4-Benzonitrile)triazolylpentyl-3-hydroxypyridine-2-one (166d)

Reaction of 164d (0.075 g, 0.197 mmol) and 1M BBr₃ (0.24 mL) in CH₂Cl₂(8 mL) within 48 h as described for the synthesis of 148a gave compound166d (43 mg, 60%) as light brown solid. ¹H NMR (400 MHz, CDCl₃) δ 7.69(d, J=8.8 Hz, 2H), 7.61 (s, 1H), 6.77 (m, 4H), 6.11 (t, J=7.1 Hz, 1H),4.37 (t, J=6.9 Hz, 2H), 3.96 (t, J=7.2 Hz, 2H), 2.99 (s, 6H), 1.99 (m,2H), 1.81 (m, 2H), 1.39 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 158.57,150.35, 148.26, 146.61, 126.84, 126.61, 118.87, 118.11, 113.72, 112.49,106.86, 49.83, 49.52, 40.46, 29.70, 28.41, 23.34. HRMS (EI) calcd forC₂₀H₂₅N₅O₂ [M]⁺ 367.200. found 367.2007.

1-(3-Benzonitrile)triazolylpentyl-3-hydroxypyridine-2-one (166e)

Reaction of 164e (0.09 g, 0.25 mmol) and 1M BBr₃ (0.30 mL) in CH₂Cl₂ (8mL) within 48 h as described for the synthesis of 148a gave compound166e (62 mg, 72%) as light brown solid. ¹H NMR (400 MHz, DMSO) δ 8.93(s, 1H), 8.76 (s, 1H), 8.01 (d, J=7.7 Hz, 2H), 7.90 (d, J=8.0 Hz, 2H),7.09 (d, J=6.4 Hz, 1H), 6.64 (d, J=7.1 Hz, 1H), 6.04 (t, J=7.0 Hz, 1H),4.41 (t, J=6.5 Hz, 2H), 3.88 (t, J=6.9 Hz, 2H), 1.89 (m, 2H), 1.67 (m,2H), 1.24 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 146.62, 145.80, 134.92,132.57, 126.69, 125.93, 120.88, 118.70, 114.06, 111.19, 107.06, 50.00,49.29, 29.43, 28.28, 23.08. HRMS (EI) calcd for C₁₉H₁₉N₅O₂ [M]⁺ 349.153.found 349.1544.

1-(4-(N,N-dimethylaniline))triazolylpentyl-3-hydroxypyridine-2-one(166f)

Reaction of 1641 (0.078 g, 0.22 mmol) and 1M BBr₃ (0.26 mL) in CH₂Cl₂ (6mL) within 48 h as described for the synthesis of 148a gave compound166f (48 mg, 64%) as light brown solid. ¹H NMR (400 MHz, CD₃OD) δ 8.39(s, 1H), 8.16 (s, 1H), 8.12 (d, J=7.5 Hz, 1H), 7.68 (d, J=7.5 Hz, 1H),7.61 (t, J=7.6 Hz, 1H), 7.04 (m, 1H), 6.76 (m, 1H), 6.21 (m, 1H), 4.45(m, 2H), 3.99 (m, 2H), 2.01 (m, 2H), 1.78 (m, 2H), 1.33 (m, 2H). ¹³C NMR(100 MHz, CDCl₃) δ 145.61, 131.97, 131.31, 129.74, 129.65, 129.10,120.30, 118.49, 112.99, 104.90, 50.07, 49.40, 29.52, 28.37, 23.17. HRMS(EI) calcd for C₁₉H₁₉N₅O₂ [M]⁺ 349.153. found 349.1539.

1-(4-(3-Trifluomethyl)-benzonitrile)triazolylpentyl-3-hydroxypyridine-2-one(166g)

Reaction of 164g (0.042 g, 0.097 mmol) and 1M BBr₃ (0.17 mL) in CH₂Cl₂(6 mL) within 48 h as described for the synthesis of 148a gave compound166g (35 mg, 85%) as light brown solid. ¹H NMR (400 MHz, CDCl₃) δ 8.24(s, 1H), 8.12 (d, J=7.9 Hz, 1H), 8.00 (s, 1H), 7.86 (d, J=7.9 Hz, 1H),6.75 (t, J=5.8 Hz, 2H), 6.12 (t, J=7.0 Hz, 1H), 4.44 (t, J=6.9 Hz, 2H),3.96 (t, J=7.1 Hz, 2H), 2.02 (m, 2H), 1.81 (m, 2H), 1.37 (m, 2H). ¹³CNMR (100 MHz, CDCl₃) δ 158.58, 146.54, 144.71, 135.49, 135.26, 128.57,126.70, 123.59, 123.54, 121.46, 115.47, 113.82, 108.63, 107.02, 50.19,49.31, 29.45, 28.30, 23.12. HRMS (EI) calcd for C₂₀H₁₈N₅O₂F₃ [M]⁺417.141. found 417.1425. HRMS (EI) calcd for C₂₀H₁₈N₅O₂F₃ [M]⁺ 417.141.found 417.1425.

1-(4-Tolyl)triazolylpentyl-3-hydroxypyridine-2-thione (167a)

Reaction of 165a (0.075 g, 0.203 mmol) and 1M BBr₃ (0.24 mL) in CH₂Cl₂(8 mL) within 48 h as described for the synthesis of 148a gave compound167a (0.042 g, 62%) as olive green solid. ¹H NMR (400 MHz, CD₃OD) δ 8.18(s, 1H), 7.66 (d, J=8.0 Hz, 2H), 7.55 (d, J=5.7 Hz, 1H), 7.20 (d, J=7.7Hz, 2H), 6.97 (d, J=6.7 Hz, 1H), 4.55 (m, 2H), 4.43 (t, J=6.7 Hz, 2H),2.34 (s, 3H), 2.01 (m, 4H), 1.39 (t, J=22.5 Hz, 2H). ¹³C NMR (100 MHz,CD₃OD) δ 149.25, 139.68, 130.93, 129.13, 127.01, 122.27, 51.41, 30.95,24.51, 21.83. HRMS (EI) calcd for C₁₉H₂₂N₄OS [M]⁺ 354.151. found354.1512.

1-(3-Tolyl)triazolylpentyl-3-hydroxypyridine-2-thione (167b)

Reaction of 165b (0.10 g, 0.271 mmol) and 1M BBr₃ (0.33 mL) in CH₂Cl₂ (8mL) within 48 h as described for the synthesis of 148a gave compound167b (0.053 g, 56%) as olive green solid. ¹H NMR (400 MHz, CD₃OD) δ 8.20(s, 1H), 7.61 (s, 1H), 7.55 (m, 2H), 7.26 (t, J=7.6 Hz, 1H), 7.12 (d,J=7.5 Hz, 1H), 6.92 (m, 2H), 4.55 (m, 2H), 4.44 (t, J=6.7 Hz, 2H), 2.36(s, 3H), 2.01 (m, 4H), 1.41 (m, 2H). ¹³C NMR (100 MHz, CD₃OD) δ 149.29,140.05, 131.87, 131.78, 130.38, 130.20, 127.90, 127.63, 124.13, 122.51,51.40, 31.07, 30.93, 28.97, 24.49, 22.02. HRMS (EI) calcd for C₁₉H₂₂N₄OS[M]⁺ 354.151. found 354.1523.

1-(2-Tolyl)triazolylpentyl-3-hydroxypyridine-2-thione (167c)

Reaction of 165c (0.072 g, 0.195 mmol) and 1M BBr₃ (0.30 mL) in CH₂Cl₂(8 mL) within 48 h as described for the synthesis of 148a gave compound167c (0.046 g, 67%) as olive green solid. ¹H NMR (400 MHz, CD₃OD) δ 7.94(s, 1H), 7.61 (m, 1H), 7.47 (s, 1H), 7.21 (m, 1H), 6.97 (m, 1H), 6.76(m, 1H), 4.52 (m, 1H), 4.45 (t, J=6.8 Hz, 1H), 2.38 (s, 1H), 2.01 (m,2H), 1.43 (m, 1H). ¹³C NMR (100 MHz, CD₃OD) δ 146.74, 135.53, 130.65,129.46, 128.69, 128.21, 125.88, 122.79, 53.41, 49.83, 29.42, 27.32,22.98, 20.55. HRMS (EI) calcd for C₁₉H₂₂N₄OS [M]⁺ 354.151. found354.1509.

1-(4-Benzonitrile)triazolylpentyl-3-hydroxypyridine-2-thione (167d)

Reaction of 165d (0.10 g, 0.271 mmol) and 1M BBr₃ (0.33 mL) in CH₂Cl₂ (8mL) within 48 h as described for the synthesis of 148a gave compound167d (0.053 g, 56%) as olive green solid. ¹H NMR (400 MHz, DMSO-d₆) δ8.53 (s, 1H), 8.27 (s, 1H), 7.82 (d, J=7.1 Hz, 1H), 7.62 (d, J=8.8 Hz,2H), 7.06 (d, J=7.5 Hz, 1H), 6.93 (m, 1H), 6.77 (m, 2H), 4.52 (d, J=6.9Hz, 2H), 4.36 (t, J=7.0 Hz, 2H), 3.21 (s, 6H), 1.88 (m, 4H), 1.34 (m,2H). HRMS (EI) calcd for C₂₀H₂₅N₅OS [M]⁺ 383.178. found 383.1776.

1-(3-Benzonitrile)triazolylpentyl-3-hydroxypyridine-2-thione (167e)

Reaction of 165e (0.095 g, 0.25 mmol) and 1M BBr₃ (0.30 mL) in CH₂Cl₂ (8mL) within 48 h as described for the synthesis of 148a gave compound167e (0.055 g, 60%) as olive green solid. ¹H NMR (400 MHz, DMSO) δ 8.76(s, 1H), 8.01 (d, J=8.1 Hz, 2H), 7.87 (m, 2H), 7.65 (d, J=6.4 Hz, 1H),7.00 (m, 1H), 6.87 (d, J=8.2 Hz, 1H), 6.70 (m, 1H), 4.44 (m, 4H), 1.89(m, 4H), 1.31 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 168.23, 155.01,145.75, 134.86, 132.56, 131.00, 125.91, 121.05, 118.68, 113.76, 112.04,111.17, 57.64, 49.81, 29.31, 27.06, 22.91. HRMS (EI) calcd forC₁₉H₁₉N₅OS [M]⁺ 365.131. found 365.1313.

1-(4-(N,N-dimethylaniline)triazolylpentyl-3-hydroxypyridine-2-thione(167f)

Reaction of 165f (0.105 g, 0.25 mmol) and 1M BBr₃ (0.33 mL) in CH₂Cl₂ (8mL) within 48 h as described for the synthesis of 148a gave compound1671 (0.072 g, 66%) as olive green solid. ¹H NMR (400 MHz, CD₃OD) δ 8.38(s, 1H), 8.15 (s, 1H), 8.10 (d, J=7.9 Hz, 1H), 7.63 (m, 3H), 6.95 (d,J=7.5 Hz, 1H), 6.67 (m, 1H), 4.57 (m, 2H), 4.48 (t, J=7.0 Hz, 2H), 2.03(m, 4H), 1.43 (m, 2H). ¹³C NMR (100 MHz, CD₃OD) δ 145.34, 132.04,131.14, 129.72, 129.58, 128.63, 121.75, 112.74, 104.99, 49.79, 29.08,27.04, 22.73. HRMS (EI) calcd for C₁₉H₁₉N₅OS [M]⁺ 365.131. found365.1310.

1-(4-(3-Trifluomethyl)-benzonitrile)triazolylpentyl-3-hydroxypyridine-2-thione(167g)

Reaction of 165g (0.105 g, 0.25 mmol) and 1M BBr₃ (0.33 mL) in CH₂Cl₂ (8mL) within 48 h as described for the synthesis of 148a gave compound167g (0.072 g, 66%) as olive green solid. ¹H NMR (400 MHz, CD₃OD) δ 8.59(s, 1H), 8.35 (s, 1H), 8.21 (d, J=7.8 Hz, 1H), 8.00 (t, J=9.5 Hz, 1H),7.58 (d, J=6.2 Hz, 1H), 6.96 (d, J=7.5 Hz, 1H), 6.72 (m, 1H), 4.57 (m,4H), 2.04 (m, 4H), 1.42 (m, 2H). ¹³C NMR (100 MHz, CD₃OD) δ 146.09,137.48, 137.24, 134.79, 130.46, 125.55, 124.87, 123.60, 122.77, 116.84,109.75, 55.10, 51.63, 30.84, 28.90, 24.47. HRMS (EI) calcd forC₁₉H₁₉N₅OS [M]⁺ 365.131. found 365.1310. HRMS (EI) calcd for C₂₀H₁₃N₅OS[M]⁺ 433.1184. found 433.1189.

Example 11 Anti-Cancer Activity of Selected3-Hydroxypyridine-2-thione-based HDAC Inhibitors

Selected HDAC model inhibitors, including compound 1.68 (below), werescreened for activity against three cancer cell lines: DU 145 (androgenindependent prostate cancer known to be responsive to HDAC inhibitors),LNCaP (androgen dependent prostate cancer, sensitive to HDAC6 activity),and Jurkat (T cell leukemia overexpressing HDAC8). The IC₅₀ values forthe selected compounds are shown in Table 14 below. When calculated IC₅₀was greater than 20 μM (highest concentration tested), it is reportedas >20 μM.

TABLE 14 Anti-cancer activity of selected HDAC inhibitor models. IC₅₀HDAC1 HDAC 6 HDAC 8 (nm) (nm) (nm) DU 145 (μm) LNCaP (μm) Jurkat (μm)

NI 681 ± 110 3675 ± 1201 NI NI NI 149g NI NI 2858 ± 944  22.84 ± 3.8 16.23 ± 1.11  10.62 ± 0.97  >20 149c NI 372 ± 35  1907 ± 771  30.0 ±5.85 14.65 ± 0.84  8.95 ± 0.69 >20 149f NI 306 ± 69  3105 ± 1649 13.59 ±2.91  7.75 ± 0.73 3.19 ± 0.3  154a NI 41% 1570 ± 1067 24.02 ± 7.47 13.11 ± 1.51  4.68 ± 0.34 >20 162d NI 911 ± 173 917 ± 139 9.33 ± 0.965.43 ± 0.47 3.27 ± 0.60 162f NI 955 ± 150 1377 ± 205  17.72 ± 3.28 10.95 ± 1.92  9.04 ± 1.31 167e NI 637 ± 160 2402 ± 263  11.08 ± 2.38 4.95 ± 0.43 5.18 ± 1.18 167e NI 356 ± 72  2831 ± 520  5.03 ± 1.13 3.49 ±0.34 3.44 ± 0.57 168 NI 534 ± 185 5470 ± 1603 16.07 ± 2.24  7.80 ± 0.556.55 ± 0.91 SAHA 2.49 ± 0.2  2.31 ± 0.74 1.49 ± 0.10

Example 12 Synthesis of Macrolide HDAC Inhibitors on a Multi-Gram Scale

Synthetic strategies which permit the synthesis of macrolide HDACinhibitors one a multi-gram scale were also developed (Scheme 22).Clarithromycin or azithromycin can be N-demethylated to afforddes-N-methyl clarithromycin or azithromycin (A). Subsequent alkylationof A with 4-ethynylbenzyl methanesulfonate afforded the modified4′-ethynylbenzylclarithromycin or 4′-ethynylbenzylazithromycin (B).Copper (I) catalyzed cycloaddition of B containing an azithromycinmacrolide with a protected azidohydoxamate analog (route A), affordedthe 1,2,3-triazole linked derivatives. Subsequent deprotection with CsFgave the desired azithromycin product (C) in good yield. Copper (I)catalyzed cycloaddition of B containing a clarithromycin macrolide withan unprotected azidohydoxamate analog (route 13), afforded the1,2,3-triazole linked clarithromycin product (D) in good yield. Theyields for each step are shown in Table 15.

TABLE 15 Starting material Compound scale Yield Comments A (Azithromycinor 25 g 55% Purified by crystallization Clarithromycin) (4:1acetone-NH₄OH) B (Azithromycin) 10 g 60% Condition: K₂CO₃, CH₃CN, 80°C., 2 h; purified by pH selective (acetate buffer) extraction B(Clarithromycin) 10 g 60% Condition: Hünig's base, DMSO, 80° C., 2 h;purified by pH selective (acetate buffer) extraction followed bycrystallization (4:1 hexane-dichloromethane) C (Azithromyoin)  5 g 80%Purified by trituration (2:1 hexane-dichloromethane) D (Clarithromycin) 5 g 80% Purified by trituration (2:1 hexane-dichloromethane)

To improve the efficiency of the multi-gram synthesis, methods ofpurifying B which obviate the need for chromatography were developed.

Methods for purifying compound A containing an azithromycin macrolideare shown in Scheme 23.5 g of crude material were dissolved in 70 mL ofMeOH. 70 mL of acetate buffer (0.75 M, pH 5.5) was added, forming acolorless suspension. 70 mL of methanol was added, forming a clearsolution. The solution was extracted with mixtures of hexanes and ethylacetate, and organics were discarded. 50% aqueous NH₄OH was added to theaqueous phase, raising the pH of the solution to 8.5. The solution wasextracted with hexanes and mixtures of hexanes and ethyl acetate, andthe organic phases were collected and dried in vacu to obtain B.

Methods for purifying compound B containing a clarithromycin macrolideare shown in Scheme 24. 4 g of crude material were dissolved in 70 mL ofMeOH. 15 mL of acetate buffer (0.75 M, pH 5.5) was added, forming acolorless suspension. 50 mL of methanol was added, forming a clearsolution. The solution was extracted with mixtures of hexanes and ethylacetate, and the organic phases were collected and dried in vacuo. B wasthen purified by crystallization from a 4:1 mixture ofhexanes:dichloromethane.

We claim:
 1. A compound of Formula I or II:

wherein M represents a macrolide subunit, E is a C₁₋₆ group, optionallycontaining one or more heteroatoms, wherein the carbon atoms and/orheteroatoms are in a linear and/or cyclic arrangement, D is asubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl group, A is a linking group connected to D, Bis an alkyl, heteroalkyl, alkylaryl or alkylheteroaryl spacer group, ZBGis a Zinc Binding Group comprising a heterocyclic moiety, R₁₃ isselected from hydrogen, a substituted or unsubstituted C₁₋₆ alkyl group,a substituted or unsubstituted C₂₋₆ alkenyl group, a substituted orunsubstituted C₂₋₆ alkynyl group, a substituted or unsubstituted C₁₋₆alkanoate group, a substituted or unsubstituted C₂₋₆ carbamate group, asubstituted or unsubstituted C₂₋₆ carbonate group, a substituted orunsubstituted C₂₋₆ carbamate group, or a substituted or unsubstitutedC₂₋₆ thiocarbamate group, R₁₄ and R₁₆ is selected from hydrogen,hydroxyl, a substituted or unsubstituted C₁₋₆ alkyl group, a substitutedor unsubstituted C₂₋₆ alkenyl group, a substituted or unsubstituted C₂₋₆alkynyl group, a substituted or unsubstituted C₁₋₆ alkanoate group, asubstituted or unsubstituted C₂₋₆ carbamate group, a substituted orunsubstituted C₂₋₆ carbonate group, a substituted or unsubstituted C₂₋₆carbamate group, or a substituted or unsubstituted C₂₋₆ thiocarbamategroup, R₁₅ is hydrogen or —OR₁₇, R₁₇ is selected from a group consistingof hydrogen, a substituted or unsubstituted C₁₋₆ alkyl group, asubstituted or unsubstituted C₂₋₆ alkenyl group, a substituted orunsubstituted C₂₋₆ alkynyl group, a substituted or unsubstituted C₁₋₆alkanoate group, a substituted or unsubstituted C₂₋₆ carbamate group, asubstituted or unsubstituted C₂₋₆ carbonate group, a substituted orunsubstituted C₂₋₆ carbamate group, or a substituted or unsubstitutedC₂₋₆ thiocarbamate group.
 2. The compound of claim 1, wherein themacrolide subunit is a multi-member lactonic ring structure.
 3. Thecompound of claim 2, wherein the macrolide subunit has the structure:

wherein W is selected from —C(O)—, —C(═NOR₁₁)—, —CH(—OR₁₁)—, —NR₁₁CH₂—,—CH₂NR₁₁—, —CH(NR₁₁R₁₁)—, —C(═NNR₁₁R₁₁)—, —NR₁₁C(O)—, —C(O)NR₁₁—, and—C(═NR₁₁)—; R is selected from the group consisting of H and C₁₋₆ alkyl;R₁ is selected from the group H, halogen, —NR₁₁R₁₁, NR₁₁C(O)R₁₁, —OR₁₁,—OC(O)R₁₁, —OC(O)OR₁₁, —OC(O)NR₁₁R₁₁, —OC(O)C₁₋₆ alcyl-R₁₂, —OC(O)C₁₋₆alkyl-R₁₂, —OC(O)OC₁₋₆ alkyl-R₁₂, —OC(O)NR₁₁C₁₋₆ alkyl-R₁₂, C₁₋₆ alkyl,C₁₋₆ alkenyl, C₁₋₆ alkynyl, optionally is substituted with one or moreR₁₂ groups; R₂ is H; R₃ is selected from H, —OR₁₁, —OC₁₋₆ alkyl-R₁₂,—OC(O)R₁₁, —OC(O)C₁₋₆ alkyl-R₁₂, —OC(O)OR₁₁, —OC(O)OC₁₋₆ alkyl-R₁₂,—OC(O)NR₁₁R₁₁, —OC(O)NR₁₁C₁₋₆ alkyl-R₁₂; alternatively, R₃ is a pyranring which can be substituted as defined above in Formulae I and II; R₄is selected from H, R₁₁, —C(O)R₁₁, —C(O)OR₁₁, —C(O)NR₁₁R₁₁, —C₁₋₆alkyl-T-R₁₁, —C₂₋₆ alkenyl-T-R₁₁, and —C₂₋₆ alkynyl-T-R₁₁; alternativelyR₃ and R₄ taken together form

T is selected from —C(O)—, —C(O)O—, —C(O)NR₁₁—, —C(═NR₁₁)—, —C(═NR₁₁)O—,—C(═NR₁₁)NR₁₁—, g) —OC(O)—, —OC(O)O—, —OC(O)NR₁₁—, —NR₁₁C(O)—,—NR₁₁C(O)O—, —NR₁₁C(O)NR₁₁—, —NR₁₁C(═NR₁₁)NR₁₁—, and —S(O)_(p)—, whereinp 0-2; R₅ is selected from R₁₁, —OR₁₁, —NR₁₁R₁₁, —OC₁₋₆ alkyl-R₁₂,—C(O)R₁₁, —C(O)C₁₋₆ alkyl-R₁₂, —OC(O)R₁₁, —OC(O)G₁₋₆ alkyl-R₁₂,—OC(O)OR₁₁, —OC(O)OC₁₋₆ alkyl-R.₁₂, —OC(O)NR₁₁R₁₁, —OC(O)NR₁₁C₁₋₆alkyl-R₁₂, —C(O)C₂₋₆ alkenyl-R₁₂, and —C(O)C₂₋₆ alkynyl-R₁₂;alternatively, R₄ and R₅ taken together with the atoms to which they arebonded, form:

wherein, Q is CH or N, and R₂₃ is —OR₁₁ or R₁₁; R₆ is selected from—OR₁₁, —C₁₋₆ alkoxy-R₁₂, —C(O)R₁₁, —OC(O)R₁₁, —OC(O)OR₁₁, —OC(O)NR₁₁R₁₁,—NR₁₁R₁₁; alternatively, R₅ and R₆ taken together with the atoms towhich they are attached form a 5-membered ring by attachment to eachother through a linker selected from —OC(R₁₂)₂O—, —OC(O)O—, —OC(O)NR₁₁—,d) —NR₁₁C(O)O—, —OC(O)NOR₁₁—, —NOR₁₁C(O)O—, —OC(O)NNR₁₁R₁₁—,—NNR₁₁R₁₁C(O)O—, —OC(O)C(R₁₂)₂—, —C(R₁₂)₂C(O)O—, —OC(S)O—, —OC(S)NR₁₁—,—NR₁₁C(S)O—, n) —OC(S)NOR₁₁—, —NOR₁₁C(S)O—, —OC(S)NNR₁₁R₁₁—,—NNR₁₁R₁₁C(S)O—, —OC(S)C(R₁₂)₂—, —C(R₁₂)₂C(S)O—; alternatively, W, R₅,and R₆ taken together with the atoms to which they are attached form:

wherein J is selected from the group consisting of O, S, and NR₁₁;R_(6′) is selected from H, unsubstituted or substituted C₁₋₄ alkyl, C₂₋₄alkenyl, which can be further substituted with C₁₋₂ alkyl or one or morehalogens, C₂₋₄ alkynyl, which can be further substituted with C₁₋₂ alkylor one or more halogens, aryl or heteroaryl, which can be furthersubstituted with C₁₋₂ alkyl or one or more halogens, —C(O)H, —COOH, —CN,—COOR₁₁, —C(O)NR₁₁R₁₁, —C(O)R₁₁, —C(O)SR₁₁; alternatively R₆ and R_(6′)taken together with the atom to which they are attached to form anepoxide, a carbonyl, an olefin, or a substituted olefin, or a C₃-C₇carbocyclic, carbonate, or carbamate, wherein the nitrogen of thecarbamate can be further substituted with a C₁-C₆ alkyl; R₇ is selectedfrom C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl, optionally issubstituted with one or more R₁₂ groups; R₁₁, for each occurrence, isselected from H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₆₋₁₀saturated, unsaturated, or aromatic carbocycle, 3-12 membered saturated,unsaturated, or aromatic heterocycle containing one or more heteroatomsselected from nitrogen, oxygen, and sulfur, —C(O)—C₁₋₆ alkyl, —C(O)—C₂₋₆alkenyl, —C(O)—C₂₋₆ alkynyl, —C(O)—C₆₋₁₀ saturated, unsaturated oraromatic carbocycle, —C(O)-3-12 membered saturated, unsaturated, oraromatic heterocycle containing one or more heteroatoms selected fromnitrogen, oxygen, and sulfur, —C(O)O—C₁₋₆ allyl, —C(O)O—C₂₋₆ alkenyl,—C(O)O—C₂₋₆ alkynyl, —C(O)O—C₆₋₁₀ saturated, unsaturated, or aromaticcarbocycle, —C(O)O-3-12 membered saturated, unsaturated, or aromaticheterocycle containing one or more heteroatoms selected from the groupconsisting of nitrogen, oxygen, and sulfur, and —C(O)NR₁₃R₁₃, optionallyis substituted with one or more R₁₂ groups; and R₁₂ is selected fromR₁₄, C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, C₃₋₁₂ saturated,unsaturated, or aromatic carbocycle, 3-12 membered saturated,unsaturated, or aromatic heterocycle containing one or more heteroatomsselected from nitrogen, oxygen, and sulfur, optionally substituted withone or more substituents.
 4. The compound of claim 1, wherein D is aphenyl, biphenyl, or naphthyl group.
 5. The compound of claim 1, whereinA is an amide, —O—, or 1,2,3-triazolyl group.
 6. The compound claim 1,wherein B is an alkyl group having from 4-9 carbon atoms.
 7. Thecompound claim 1, wherein the zinc binding group is3-hydroxypyridine-2-thione.
 8. The compound claim 1, wherein thecompound is defined by the following formula

wherein R is:

and m is an integer from 1-12 inclusive.
 9. The compound of claim 8,wherein m is an integer from 5-9 inclusive.
 10. A pharmaceuticalcomposition comprising an effective amount of the compound of claim 1 incombination with a pharmaceutically acceptable diluent, excipient, orcarrier.
 11. The composition of claim 10, wherein the composition isformulated for enteral or parenteral administration.
 12. The compositionof claim 10, wherein the composition is formulated for immediaterelease, modified release, and combinations thereof.
 13. The compositionof claim 12, wherein the formulation is selected from the groupconsisting of delayed release, extended release, pulsatile release, andcombinations thereof.
 14. A method of treating a disease or disorder ina human or animal subject in need thereof comprising administering aneffective amount of a compound claim 1 to the subject, wherein thedisease or disorder is selected from the group consisting of cancer,inflammation, and parasitic infections.
 15. The method of claim 14,wherein the disease or disorder to be treated is cancer.
 16. The methodof claim 15, wherein the cancer is selected from the group consisting oflung cancer, myeloma, leukemia, lymphoma, breast cancer, prostatecancer, pancreatic cancer, cervical cancer, ovarian cancer, and livercancer.
 17. The method of claim 14, wherein the parasitic infection isselected from the group consisting of malaria and leishmaniasis.